Glucose Transport Mutants For Production Of Biomaterial

ABSTRACT

A method is disclosed for restoring a Glu +  phenotype to a PTS − /Glu −  bacterial cell which was originally capable of utilizing a phosphotransferase transport system (PTS) for carbohydrate transport. Bacterial cells comprising the Glu +  phenotype have modified endogenous chromosomal regulatory regions which are operably linked to polynucleotides encoding galactose permeases and glucokinases.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to application PCT/US03/31544, filedOct. 3, 2003, U.S. Provisional Application 60/416,166 filed Oct. 4, 2002and U.S. Provisional Application 60/374,931 filed Oct. 4, 2002, whichare hereby incorporated by reference.

TECHNICAL FIELD

The present invention relates to genetically engineering metabolicpathways in bacterial host cells and provides methods and systems forthe production of desired products in the engineered host cells. Inparticular, the invention relates to the enhancement of glucosetransport in host strains, which were originally capable of using aphosphoenolpyruvate (PEP):phosphotransferase transport system (PTS) forglucose transport, by reducing PTS phosphoenolpyruvate (PEP) consumptionand redirecting PEP or PEP precursors into a desired metabolic pathway,such as the common aromatic amino acid pathway.

BACKGROUND ART

Many industrially important microorganisms use glucose as their maincarbon source to produce biosynthetic products. Therefore,cost-effective and efficient biosynthetic production of these productsrequire that a carbon source, such as glucose be converted to saidproducts at a high percentage yield. To meet this need, it would beadvantageous to increase the influx of carbon sources into and throughvarious metabolic pathways, such as the common aromatic pathway, thetricarboxylic acid (TCA) pathway, and the anaplerotic oxaloacetatesynthetic pathway.

In the initial stage of host cell carbohydrate metabolism, each glucosemolecule is converted to two molecules of phosphoenolpyruvate (PEP) inthe cytosol. PEP is one of the major metabolic building blocks thatcells use in their biosynthetic as routes. For example, PEP may befurther converted to pyruvate and chemical reactions that convertglucose to pyruvate are referred to as the Embden-Meyerhoff pathway. Allof the metabolic intermediates between the initial glucose carbohydrateand the final product, pyruvate, are phosphorylated compounds. Bacteria,which ferment glucose through the Embden-Meyerhof pathway, such asmembers of Enterobacteriacea and Vibrionaceae, are described in Bouvetet al., (1989) International Journal of Systematic Bacteriology,39:61-67. Pyruvate may then by metabolized to yield products such aslactate, ethanol, formate, acetate and acetyl CoA. (See FIGS. 1A and1B).

In addition to the Embden-Meyerhof pathway, many bacteria posses anactive transport system known as the phosphoenolpyruvate (PEP)—dependentphosphotransferase transport system (PTS). This system couples thetransport of a carbon source, such as glucose to its phosphorylation.The phosphoryl group is transferred sequentially from PEP to enzyme Iand from enzyme I to protein HPr. The actual translocation step iscatalyzed by a family of membrane bound enzymes (called enzyme II), eachof which is specific for one or a few carbon sources. Reference is madeto Postma et al., (1993) Phosphoenolpyruvate: CarbohydratePhosphotransferase Systems in Bacteria, Microbiol. Reviews. 57:543-594and Postma P. W. (1996) Phosphotransferase System for Glucose and OtherSugars. In: Neidhardt et al., Eds. Escherichia coli AND Salmonellatyphimurium: CELLULAR AND MOLECULAR BIOLOGY. Vol. 1. Washington, D.C.ASM Press pp 127-141. However, due to the fact that PTS metabolizes PEPto phosphorylate the carbon source, the PTS system decreases theefficiency of carbon substrate conversion to a desired product. Inglycolysis, two molecules of PEP are formed for every molecule ofglucose catabolized. However, one molecule of PEP is required for PTS tofunction, leaving only one molecule of PEP available for otherbiosynthetic reactions.

Due to the role of PEP as a central metabolite, numerous approaches havebeen utilized to increase PEP supply in the cell and some of these arelisted below:

a) eliminating pyruvate kinase activity by producing pyk mutants.Pyruvate kinase catalyzes the conversion of PEP to pyruvate. (Mori etal., (1987) Agric. Biol. Chem. 51:129-138);

b) eliminating PEP carboxylase activity by producing ppc mutants. PEPcarboxylase catalyzes the conversion of PEP to oxaloacetate. (Miller etal., (1987) J. Ind. Microbiol. 2:143-149);

c) amplifying the expression of pps which encodes PEP synthase. PEPsynthase catalyzes the conversion of pyruvate to PEP (U.S. Ser. No.08/307,371); and

d) increasing the supply of D-erythrose-4-phosphate (E4P) by for exampleoverexpression of a transketolase gene (tktA or tktB) (U.S. Pat. No.5,168,056) or overexpression of the transaldolase gene (talA) (lida etal., (1983) J. Bacteriol. 175:5375-5383). Transketolase catalyzes theconversion of D-fructose-6-phosphate to E4P and transaldolase catalyzesthe conversion of D-sedoheptulose-7-phosphate plusglyceraldehyde-3-phosphate to E4P plus fructose-6-phophate.

In addition to the above listed approaches, researchers have looked atmethods of decreasing PTS:PEP dependent consumption by eliminating ormodifying the function of the PTS. This approach is also attractivebecause PEP is twice as energetic as ATP. Many of these efforts focus onusing an inactive PTS system. Examples of studies manipulating the PTSsystem include

-   -   a) restoring a glucose phenotype (Glu⁺) in PTS inactivated E.        coli cells by introducing the genes glf and glk which encode a        glucose-facilitated diffusion protein and glucokinase,        respectively, from Zymomonas mobilis, wherein the E. coli cells        have an inactivated PTS due to a deletion of the pstHlcrr operon        (U.S. Pat. No. 5,602,030 and Snoep et al., (1994) J. Bact.        176:2133-2135) and    -   b) subjecting PTS⁻/Glu⁻ E. coli strains to continuous culture        selection on glucose and obtaining Glu+ revertants (PTS⁻/Glu⁺)        with the capacity to obtain growth rates similar or higher than        that of wild-type PTS⁻/Glu⁻ strains. (Flores et al., (2002)        Metab. Eng 4:124-137; Flores et al., (1996) Nature Biotechnol.        14:620-623 and WO96/34961).

However, these approaches have various limitations. In general, the useof heterologous genes does not always work efficiently in new hosts.Additionally, membrane proteins, such as a glucose-facilitated diffusionprotein, are usually intimately associated with lipids in the cellmembrane and these can vary from species to species. Introduced solubleproteins such as glucokinase, may be subject to protease degradation.Further the use of spontaneous mutations in a cell to regain a phenotypecan have unpredictable outcomes, and for industrial processes it isdesirable to use completely characterized strains.

Contrary to the methods previously described, the present inventionincreases carbon flow to metabolic pathways in bacterial strains capableof transporting glucose without consuming PEP during the process. Theconserved PEP or PEP precursors can then be redirected into a givenmetabolic pathway for enhanced production of a desired product. Thesestrains are generated in cells having an inactivated PEP-dependent PTSby modifying an endogenous chromosomal regulatory region that isoperably linked to a glucose assimilation protein and more specificallyto a glucose transporter and/or a glucose phosphorylating protein, torestore or re-attain the ability of the cell to use glucose as a carbonsource while maintaining an inactivated PTS. These cells are designatedPTS⁻/Glu⁺.

SUMMARY OF THE INVENTION

Accordingly, there is provided by the present invention a method forincreasing carbon flow into a metabolic pathway of a bacterial host cellwherein the host cell was originally capable of utilizing a PTS forcarbohydrate transport. The method comprises selecting a bacterial hostcell which is phenotypically PTS⁻/Glu⁻ and modifying an endogenouschromosomal regulatory region which is operably linked to a nucleic acidencoding a polypeptide involved in glucose assimilation to restore theGlu⁺ phenotype.

In a first aspect, the invention pertains to a method of increasingcarbon flow into a metabolic pathway of a PTS⁻/Glu⁻ bacterial host cellwhich was originally capable of utilizing a phosphotransferase transportsystem (PTS) for carbohydrate transport which comprises a) modifying anendogenous chromosomal regulatory region which is operably linked to anucleic acid encoding a glucose assimilation protein in a PTS⁻/Glu⁻ hostcell by transforming the PTS⁻/Glu⁻ host cell with a DNA constructcomprising a promoter and DNA flanking sequences corresponding toupstream (5′) regions of the glucose assimilation protein; b) allowingintegration of the DNA construct to restore a Glu+ phenotype; and c)culturing the transformed host cell under suitable culture conditions,wherein the carbon flow into a metabolic pathway of the transformed hostcell is increased compared to the carbon flow into the same metabolicpathway in a corresponding PTS bacterial host cell cultured underessentially the same culture conditions. In one embodiment of the methodthe promoter is a non-host cell promoter or a modified endogenouspromoter. In a second embodiment the glucose assimilation protein is aglucose transporter, preferably a galactose permease obtained from E.coli or a glucose transporter having at least 80% sequence identitythereto. In a third embodiment the glucose assimilation protein is aphosphorylating protein, preferably a glucokinase obtained from E. colior a glucokinase having at least 80% sequence identity thereto. In afourth embodiment of the method the bacterial host cell is selected fromthe group consisting of E. coli cells, Bacillus cells and Pantoea cells.In a fifth embodiment, the PTS⁻/Glu⁻ host cell is obtained from a PTScell by deletion of one or more genes selected from the group consistingof ptsI, ptsH and crr. In a sixth embodiment, the PTS⁻/Glu⁺ host cell istransformed with a polynucleotide encoding a protein selected from thegroup consisting of a transketolase, a transaldolase, aphosphoenolpyruvate synthase, DAHP syntheses DHQ synthase, DHQdehydratase, shikimate dehydrogenase, shikimate kinase EPSP synthase andchorismate synthase.

In a second aspect, the invention pertains to a method as described inthe first aspect and further comprising modifying an endogenouschromosomal regulatory region which is operably linked to a nucleic acidencoding a glucokinase in the PTS⁻/Glu⁻ host cell by transforming thePTS⁻/Glu⁻ host cell with a second DNA construct comprising a promoterand DNA flanking sequences corresponding to upstream (5′) regions of theglucokinase.

In a third aspect, the invention pertains to a method for increasing theproduction of a desired product in a PTS⁻/Glu⁻ bacterial host celloriginally capable of utilizing a PTS for carbohydrate transport whichcomprises a) transforming a bacterial host cell having a PTS⁻/Glu⁻phenotype with a DNA construct comprising a promoter, wherein said DNAconstruct is chromosomally integrated into the PTS⁻/Glu⁻ host cellreplacing an endogenous promoter which is operably linked to a nucleicacid encoding a glucose assimilation protein; b) culturing thetransformed bacterial host cell under suitable conditions; c) allowingexpression of the glucose assimilation protein to obtain a host cellhaving a PTS⁻/Glu⁺ phenotype, and d) obtaining an increased amount of adesired product in the transformed bacterial host cell compared to theamount of the desired product produced in a corresponding PTS bacterialcell cultured under essentially the same culture conditions, whereinsaid desired product is selected from the group consisting of pyruvate,PEP, lactate, acetate, glycerol, succinate, ethanol and chorismate. Inone embodiment the host cell is selected from the group consisting of E.coli cells, Bacillus cells and Pantoea cells. In a second embodiment theglucose assimilation protein is a galactose permease obtained from E.coli or a glucose transporter having at least 80% sequence identitythereto. In a third embodiment, the glucose assimilation protein is aglucokinase obtained from E. coli or a glucokinase having at least 70%sequence identity thereto.

In a fourth aspect, the invention pertains to a method of increasingcarbon flow into a metabolic pathway of a PTS⁻/Glu⁻ bacterial host celloriginally capable of utilizing a phosphotransferase transport system(PTS) for carbohydrate transport which comprises a) modifying anendogenous chromosomal regulatory region which is operably linked to anucleic acid encoding a galactose permease in a PTS⁻/Glu⁻ host cell bytransforming the PTS⁻/Glu⁻ host cell with a first DNA constructcomprising a promoter and DNA flanking sequences corresponding toupstream (5′) regions of the galactose permease; b) modifying anendogenous chromosomal regulatory region which is operably linked to anucleic acid encoding a glucokinase in the PTS⁻/Glu⁻ host cell bytransforming the PTS⁻/Glu⁻ host cell with a second DNA constructcomprising a promoter and DNA flanking sequences corresponding toupstream (5′) regions of the glucokinase; c) allowing integration of thefirst and the second DNA constructs, wherein the first DNA constructreplaces an endogenous promoter of the nucleic acid encoding thegalactose permease and the second DNA construct replaces an endogenouspromoter of the nucleic acid encoding the glucokinase wherein both thegalactose permease and the glucokinase are expressed in the host celland wherein said expression results in an increase in carbon flow into ametabolic pathway of the transformed host cell compared to carbon flowinto the same metabolic pathway in the corresponding unaltered PTS⁻/Glu⁻bacterial cell. In one embodiment the metabolic pathway is the commonaromatic pathway. In a second embodiment the method further comprisestransforming the PTS⁻/Glu⁻ host cell with a polynucleotide encoding aprotein selected from the group consisting of a transketolase, atransaldolase and a phosphoenolpyruvate synthase.

In a fifth aspect, the invention pertains to a method of restoring aGlu+ phenotype to a PTS⁻/Glu⁻ bacterial host cell which was originallycapable of utilizing a phosphotransferase transport system (PTS) forcarbohydrate transport which comprises a) modifying an endogenouschromosomal regulatory region which is operably linked to a nucleic acidencoding a glucose transporter in a PTS⁻/Glu⁻ host cell by transformingthe PTS⁻/Glu⁻ host cell with a first DNA construct comprising a promoterand DNA flanking sequences corresponding to upstream (5′) regions of theglucose transporter; b) allowing integration of the first DNA construct,wherein the first DNA construct replaces an endogenous promoter of thenucleic acid encoding the glucose transporter; and c) allowingexpression of the glucose transporter, wherein said expression restoresa Glu+ phenotype to the PTS⁻/Glu⁻ host cell. In a preferred embodimentthe method according to this aspect further comprises modifying anendogenous chromosomal regulatory region which is operably linked to anucleic acid encoding a glucokinase in the PTS⁻/Glu⁻ host cell bytransforming the PTS⁻/Glu⁻ host cell with a second DNA constructcomprising an exogenous promoter and DNA flanking sequencescorresponding to upstream (5′) regions of the glucokinase; allowingintegration of the second DNA construct wherein the second DNA constructreplaces an endogenous promoter of the nucleic acid encoding theglucokinase; and allowing expression of the glucokinase. In oneembodiment the restored Glu⁺ cells have a specific growth rate of atleast about 0.4 hr⁻¹. In another embodiment the glucose transporter is agalactose permease.

In a sixth aspect the invention pertains to a method of increasingphosphoenolpyruvate (PEP) availability in a bacterial host cell whichcomprises a) selecting a bacterial host cell having a PTS⁻/Glu⁻phenotype, wherein the bacterial host was originally capable ofutilizing a phosphotransferase transport system (PTS) for carbohydratetransport; b) modifying an endogenous chromosomal regulatory sequence ofthe selected bacterial host cell comprising transforming said selectedbacterial host cell with a DNA construct comprising a promoter, whereinsaid DNA construct is chromosomally integrated into the selectedbacterial host cell replacing an endogenous promoter which is operablylinked to a nucleic acid encoding a glucose assimilation protein; c)culturing the transformed bacterial host cell under suitable conditions;and d) allowing expression of the glucose assimilation protein to obtainan altered host cell having a PTS⁻/Glu⁺ phenotype, wherein the PEPavailability is increased compared to the PEP availability in acorresponding unaltered PTS bacterial host cell cultured underessentially the same culture is conditions. In one embodiment theglucose assimilation protein is a galactose permease and the DNAconstruct comprises an exogenous promoter which replaces the endogenouspromoter of the galactose permease. In another embodiment the glucoseassimilation protein is a glucokinase and the DNA construct comprises anexogenous promoter which replaces the endogenous promoter of aglucokinase.

In an eighth aspect, the invention pertains to a method for increasingthe growth rate of a PTS⁻/Glu⁻ bacterial host cell originally capable ofutilizing a phosphotransferase transport system (PTS) for carbohydratetransport which comprises, a) modifying an endogenous chromosomalregulatory region which is operably linked to a nucleic acid encoding agalactose permease in a PTS⁻/Glu⁻ host cell by transforming thePTS⁻/Glu⁻ host cell with a first DNA construct comprising an exogenouspromoter and DNA flanking sequences corresponding to (5′) upstreamregion of the galactose permease; b) modifying an endogenous regulatoryregion which is operably linked to a nucleic acid encoding a glucokinasein the PTS⁻/Glu⁻ host cell by transforming the PTS⁻/Glu⁻ host cell witha second DNA construct comprising an exogenous promoter and DNA flankingsequences corresponding to upstream (5′) regions of the glucokinase; c)allowing integration of the first and the second DNA constructs, whereinthe first DNA construct replaces the endogenous promoter of the nucleicacid encoding the galactose permease and the second DNA constructreplaces the endogenous promoter of the nucleic acid encoding theglucokinase d) culturing the transformed bacterial host cell undersuitable conditions; and e) allowing expression of the galactosepermease and the glucokinase from the modified regulatory regions toobtain an altered bacterial cell having an increase specific growth ratecompared to the specific growth rate of a corresponding unaltered PTSbacterial host cell cultured under essentially the same cultureconditions.

In a ninth aspect, the invention pertains to a method for increasing theproduction of a desired product in a PTS⁻/Glu⁻ E. coli host celloriginally capable of utilizing a PTS for carbohydrate transport whichcomprises a) modifying an endogenous chromosomal regulatory region whichis operably linked to a nucleic acid encoding a galactose permease in anE. coli PTS⁻/Glu⁻ cell by transforming the E. coli PTS⁻/Glu⁻ cell with afirst DNA construct comprising an exogenous promoter and DNA flankingsequences corresponding to upstream (5′) regions of the galactosepermease; b) modifying an endogenous chromosomal regulatory region whichis operably linked to a nucleic acid encoding a glucokinase in the E.coli PTS⁻/Glu⁻ cell by transforming the E. coli PTS⁻/Glu⁻ cell with asecond DNA construct comprising an exogenous promoter and DNA flankingsequences corresponding to upstream (5′) regions of the glucokinase; c)culturing the transformed E. coli PTS⁻/Glu⁻ cell under suitableconditions to allow expression of the galactose permease and expressionof the glucokinase; and d) obtaining an increased amount of a desiredproduct in the transformed E. coli cells compared to the amount of thedesired product in a corresponding PTS⁻/Glu⁻ E. coli cell cultured underessentially the same culture conditions wherein the desired product isethanol, chorismate or succinate.

In a tenth aspect, the invention pertains to the transformed bacterialcells obtained according to the methods of the first through ninthaspects. In one preferred embodiment, the transformed bacterial cellsare E. coli cells, Bacillus cells or Pantoea cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B illustrate the pathways of central carbon metabolism inE. coli, showing derivation of the carbon skeletons for various desiredcompounds including compounds in the biosynthesis of amino acids. FromFIG. 1A it can be observed that (a) glucose is transported across thecell membrane by a galactose permease (GalP) transport protein and (b)that glucose moves across the membrane as to be phosphorylated by PEP inthe PTS system. The phosphorylation of glucose by the PTS is the majorconsumer of PEP in PTS cells and the percentages shown in the figurerepresent the amount of PEP channeled into competing pathways asdescribed by Holms (1986) The central metabolic pathways of Escherichiacoli: relationship between flux and control at a branch point,efficiency of conversion to biomass, and excretion of acetate. In:CURRENT TOPICS IN CELLULAR REGULATION, Vol. 28, pp. 69-105 AcademicPress, New York. For example, 66% of the PEP produced is used in the PTSsystem. The following metabolic systems are schematically illustrated inthe figure: Embden-Meyerhoff pathway (glycolysis), the pentose phosphatepathway, tricarboxylic acid (TCA) pathway, common aromatic pathway, andthe Entner-Doudoroff pathway.

The following abbreviations are used in the figure and throughout thedisclosure: PEP=phosphoenolpyruvate;DAHP=3-deoxy-D-arabino-heptulosonate 7-phosphate; DHQ=3-dehydroquinate;DHS=3-dehydroshikimate; SHK=shikimate; S3P=shikimate 3-phosphate;EPSP=5-enolpyruvyl shikimate 3-phosphate; PHE=phenylalanine;TYR=tyrosine; TRP=tryptophan; Pyk=pyruvate kinase, which is encoded bythe gene pyk; and Ppc=PEP carboxylase, which is encoded by the gene ppc.Further, the following genes are illustrated for the common aromaticpathway: aroB which encodes DHQ synthase; aroD which encodes DHQdehydratase; aroE which encodes shikimate dehydrogenase; aroL and aroKwhich encode shikimate kinase; aroA which encodes EPSP synthase and aroCwhich encodes chorismate synthase. While not specifically illustrated,one skilled in the art is aware that aroG, aroF and aroH encode thethree isozymes of DAHP synthase which catalyzes the conversion ofErythrose-4P (E4P) and PEP to DAHP in E. coli. FIG. 1B illustrates thevaried compounds, the production of which, may be enhanced by anincrease in carbon flux and PEP availability according to the methodsencompassed by the invention.

FIG. 2 illustrates the DNA sequence of the GalP-ptrc DNA cassette as setforth in SEQ ID NO. 1 cloned into the R6K vector (creating pR6K-galP).Italics and bold nucleotide sequences represent the loxP sequences; boldand underlined nucleotide sequences represent the chloramphenicolacetyltransferase (CAT) gene in reverse orientation to galP; underlinedsequences represent the −35 region (TTGACA) and the −10 region (TATAAT)of the trc promoter; the italicized nucleotides represent the lacoperator of the trc promoter; and the bold nucleotides represent thegalP ATG start codon. (Reference is made to example 1B).

FIG. 3 illustrates the DNA sequences of the galP-trc DNA cassette afterremoval of the CAT gene (SEQ ID NO. 2). The bold nucleotides representthe galP upstream sequence; italicized nucleotides represent a singleloxP site, and bold and italics nucleotides represent the trc promoterregion, wherein the −35 box and the −10 box are underlined. (Referenceis made to example 1E).

FIG. 4 illustrates the DNA sequence of the glk-trc DNA cassette as setforth in SEQ ID NO. 3 and which was used to clone into the R6K vector.Italics and bold nucleotide sequences represent the loxP sequences; boldand underlined nucleotide sequences represent the CAT gene in reverseorientation to glk; underlined sequences represent the −35 region(CTGACA) and the −10 region (TATAAT) of a trc derivative promoter; theitalicized nucleotides represent the lac operator of the trc promoter;and the bold nucleotides represent the glk ATG start codon. (Referenceis made to example 1F).

FIG. 5 illustrates a plasmid map of pMCGG and reference is made toexample 1G. The galP and glk open reading frames (orfs) were cloned intopACYC177, each under the control of the trc promoter. Trc=trc promoter;galP=galactose permease coding sequence; glk=glucokinase codingsequence; KmR′=kanamycin resistance marker of pACYC177 (interrupted bycloned genes); Amp=ampicillin resistance marker of pACYC177 andori=plasmid origin of replication.

FIG. 6 illustrates a plasmid map of pTrcm42 and reference is made toexample 1A. LoxP=loxP sites; trc=trc promoter, laclq=gene encoding theLacl^(q) repressor protein; CAT=CAT encoding gene; 5S=5StRNA; rrnBT1 and2=ribosomal RNA terminators; Amp=ampicillin resistance gene of pTrc99a;and ori=pMB1origin of replication.

FIGS. 7A and 7B. FIG. 7A illustrates a diagram of a schematic promoterDNA integration cassette comprising loxP-CAT-loxP-ptrc. DNA homology toa desired site of integration is added by PCR such that it flanks theentire cassette and reference is made to example 1E and 1F. loxP=loxPsites; CAT=chloramphenicol acetyltransferaces gene, trc=trc promoter;and ATG=start codon of the glucose assimilation gene targeted forintegration of the DNA cassette. FIG. 7B illustrates a plasmid map ofpRGK-galP-trc, which was created by amplifying the DNA cassette of FIG.7A flanked by 40-bp of homology to the regulatory region of galP from221-183 bp upstream of the ATG start codon of galP (5′ flank) and 40 bpupstream of and including the ATG start codon of galP (3′ flank) whereinploxP=plasmid encoded loxP; loxP=introduced loxP sequences from the DNAcassette; trc=trc promoter; Km=kanamycin resistance gene; CAT is asdefined above and ori=R6K plasmid origin of replication.

FIG. 8 illustrates the nucleotide sequence of plasmid pSYCO101 (SEQ IDNO. 4).

FIG. 9 depicts a plasmid map of pSYCO101, wherein DAR1 (dihydroxyacetonephosphate reductase) and GPP2 (glycerol-phosphate phosphatase) areglycerol pathway genes; STR(R) is a spectinomycin resistance encodinggene; pSC101 ori is an origin of replication of the plasmid; AspA termis an aspartate ammonia lyase gene terminator; dhaB1-3, dhaX, and orf W,X, Y are 1, 3 propanediol pathway genes; Thr atten is an E. colithreonine operon attenuator; TonB term is an E. coli tonB geneterminator; trc is the trc promoter and EcoR1 is the EcoR1 restrictionenzyme sites in pSYCO101. (Reference is made to example 1H).

FIGS. 10A and 10B illustrate cell growth and the production of glyceroland 1,3-propanediol over fermentation time (h) in a PTS⁻/Glu⁺ E. coliwith a plasmid encoded PEP-independent glucose transport system. ThePTS⁻/Glu⁻ strain, KLpts7 (example 1D) was transformed with pMCGG(example 1G) and with pSYCO101 and the resultant strain was tested andanalyzed for cell growth, glycerol and 1,3-propanediol production.Optical density (OD₆₀₀) is represented by -♦-, glycerol concentration isrepresented by -▴-, and 1,3-propanediol concentration is represented by-▪-.

FIGS. 11A and 11B illustrate cell growth and the production of glyceroland 1,3-propanediol over fermentation time (h) in a PTS⁻/Glu⁺ E. coliwith the expression of galP controlled by a chromosomally integrated trcpromoter and that of glk by natural regulation. The PTS⁻/Glu⁻, KLpts7(example 1D) was made Glu⁺ by integration of the trc promoter at thegalP target site to yield strain KlgalP-ptrc (example 1E). This strainwas transformed with pSYCO103 and analyzed by fermentation (example 2).The parameters examined include, for FIG. 11A cell density (OD₆₀₀) (-♦-)and for FIG. 11B glycerol concentration (-▴-) and 1,3-propanediolconcentration (-▪-).

FIGS. 12A and 12B illustrate cell growth and the production of glyceroland 1,3-propanediol over fermentation time (h) in a PTS⁻/Glu⁺ E. coliwith the expression of galP and glk controlled by a chromosomallyintegrated trc promoter. The PTS⁻/Glu⁻, KLpts7 (example 1D) was madeGlu+ by integration of the trc promoter at the galP and glk target sitesto yield strain KLGG (example 1F). This strain was transformed withpSYCO101 and analyzed by fermentation (example 3). The parametersexamined include, for FIG. 12A cell density (OD₆₀₀) (-♦-) and for FIG.12B glycerol concentration (-▴-) and 1,3-propanediol concentration(-▪-).

FIG. 13 is a map of plasmid pVHGalPglk11 as described in example 1G.

FIG. 14A-E illustrates the nucleotide sequences (SEQ ID NO. 25) andcoding sequences for galP and Glk (SEQ ID NO. 26) of pVHGalPglk11.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, and biochemistry,which are within the skill of the art. Such techniques are explainedfully in the literature, such as, MOLECULAR CLONING: A LABORATORYMANUAL, second edition (Sambrook et al., 1989) Cold Spring HarborLaboratory Press; CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubelet al., eds., 1987 and annual updates); GENE EXPRESSION TECHNOLOGY,(Goeddel, D. ed., 1991, METHODS IN ENZYMOLOGY Vol. 185 Academic Press,San Diego, Calif.); GUIDE TO PROTEIN PURIFICATION (Deutshcer M. P. ed.,1989, METHODS IN ENZYMOLOGY, Academic Press, San Diego, Calif.); PCRPROTOCOLS: A GUIDE TO METHODS AND APPLICATIONS (Innis et al., 1990,Academic Press, San Diego, Calif.); OLIGONUCLEOTIDE SYNTHESIS (M. J.Gait, ed., 1984); PCR: THE POLYMERASE CHAIN REACTION, (Mullis et al.,eds., 1994); MANUAL OF INDUSTRIAL MICROBIOLOGY AND BIOTECHNOLOGY, SecondEdition (A. L. Demain, et al., eds. 1999); MANUAL OF METHODS FOR GENERALBACTERIOLOGY (Phillipp Gerhardt, R. G. E. Murray, Ralph N. Costilow,Eugene W. Nester, Willis A. Wood, Noel R. Krieg and G. Briggs Phillips,eds), pp. 210-213 American Society for Microbiology, Washington, D.C.;and BIOTECHNOLOGY: A TEXTBOOK OF INDUSTRIAL MICROBIOLOGY, (Thomas D.Brock) Second Edition (1989) Sinauer Associates, Inc., Sunderland, Mass.

DEFINITIONS

Unless defined otherwise herein, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill so in the art to which this invention pertains. Singleton, et al.,DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2D ED., John Wiley andSons, New York (1994) and Hale and Marham, THE HARPER DICTIONARY OFBIOLOGY, Harper Perennial, New York (1991) provide one of skill withgeneral dictionaries of many of the terms used in this invention.

Numeric ranges are inclusive of the numbers defining the range. Unlessotherwise indicated nucleic acids are written left to right in the 5′ to3′ orientation and amino acid sequences are written left to right inamino to carboxy orientation, respectively.

The headings provided herein are not limitations of the various aspectsor embodiments of the invention which can be had by reference to thespecification as a whole. Accordingly, the terms defined immediatelybelow are more fully defined by reference to the specification as awhole.

The references, issued patents and pending patent applications citedherein are incorporated by reference into this disclosure.

The phosphotransferase system (PTS) (E.C. 2.7.1.69) refers to thephosphoenolpyruvate-dependent sugar uptake system. This is a system oftransporter proteins that participate in transport andphosphoenolpyruvate dependent phosphorylation of several sugars. Thesystem is comprised of two phosphoproteins, enzyme I and HPr, which arecommon to all PTS carbohydrates and catalyze the transfer of aphosphoryl group from PEP to the carbohydrate specific membrane boundenzyme II and then to the carbohydrate. In some cases enzyme III ispositioned between HPr and enzyme II. To distinguish the various enzymesII and enzymes III, a three letter superscript is used to indicate whichcarbohydrate is the preferred substrate. For example Enzyme II^(glc)means glucose is the preferred substrate. However other substrates maybe used.

The terms “PtsI” and “Enzyme I” refer to the phosphotransferase EC2.7.3.9 encoded by ptsI in E. coli. The terms “HPr” and “PtsH” refer tothe phosphocarrier protein encoded by ptsH in E. coli. The terms“glucose-specific IIA component”, “Enzyme II^(glc)” and “Crr” refer toEC 2.7.1.69 encoded by err in E. coli. The PTS comprises PtsI, PtsH andCrr and functionally equivalent proteins.

The terms “PTS⁻/Glu⁻ phenotype” and “PTS⁻/Glu⁻” mean a host cell whichhas a significantly reduced ability to utilize glucose as a carbonsource because the PEP-dependent PTS is inactivated compared to thecorresponding wild-type PTS cell. Effectively, less PEP is utilized totransport glucose than in the wild-type PTS cell.

“Restoring the glucose⁺ (Glu⁺) phenotype” means a host cell capable ofusing glucose as a carbon source despite the inactivation of the PTS.Further a “PTS⁻/Glu⁺ phenotype” as used herein means a PTS⁻/Glu⁻ hostcell that has a restored Glu⁺ phenotype.

“Increased phosphoenolpyruvate (PEP) availability” means increasing theamount of intracellular PEP which enhances carbon committed to ametabolic or productive pathway, said PEP which would otherwise havebeen metabolized in the PTS for phosphorylation of glucose.

The phrase “increasing carbon flow” means increasing the availability ofcarbon substrates to metabolic or productive pathways, said carbonsubstrate which would otherwise be diverted by the metabolism of PEP inthe PTS. Carbon flow to a particular pathway can be measured by wellknow methods such as gas chromatography and mass spectroscopy. Carbonflow as measured by produced product may be at least 2%, 5%, 10%, 15%,20%, 25%, 30%, or greater than the carbon flow in a corresponding PTScell grown under essentially the same growth conditions.

The term “specific growth rate (μ)” refers to the increase of mass orcell number per time. In one embodiment of the invention a cell having arestored Glu⁺ phenotype will have a specific growth rate (μ) of about atleast 0.3 hr¹, at least 0.4 hr⁻¹, at least 0.5 hr¹, at least 0.6 hr⁻¹,at least 0.7 hr⁻¹ and at least 0.8 hr⁻¹ when grown on is glucose.

The terms “regulatory region” and “regulatory sequence” are usedinterchangeably herein and mean a nucleic acid sequence that is operablylinked with a coding region of a gene to effect expression of the codingregion. A regulatory sequence can inhibit, repress or promote expressionof the operably linked coding sequence or translation of the mRNA.Examples of regulatory sequences include promoters, enhancers, ribosomebinding sites, operators and silencers.

An “endogenous chromosomal regulatory region” and “homologouschromosomal regulatory region” refer to a chromosomal regulatory region,which naturally occurs in a host cell and which is operably linked to apolynucleotide encoding a glucose assimilation protein in said hostcell.

The term “promoter” as used herein refers to a regulatory nucleic acidsequence that functions to direct transcription of a downstream gene orgenes. A promoter according to the invention comprises two consensusregions. The first consensus region is centered about 10 base pairs (bp)upstream from the start site of transcription initiation and is referredto as the −10 consensus region (also the −10 box or Pribnow box). Thesecond consensus region is centered about 35 bp upstream of the startsite and is referred to as the −35 consensus box or sequence. A linkeror spacer sequence is positioned between the consensus boxes andgenerally comprises 14 to 20 bp.

An “exogenous promoter” as used herein is a promoter, other than anaturally occurring promoter, which is operably linked to an endogenouscoding region of a glucose assimilation protein of interest in a hostcell and includes but is not limited to non-native promoters, syntheticpromoters, and modified naturally occurring promoters. Modifiednaturally occurring promoters include native endogenous promoters whichare operably linked to a polynucleotide encoding a glucose assimilationprotein, wherein the native promoter has been altered and thenreintroduced into a host cell chromosome and include native endogenouspromoters which are not operably linked to an endogenous coding regionof a glucose assimilation protein.

The terms “derivative promoter”, “modified promoter” and “variantpromoter” mean a promoter, wherein at least one nucleotide of thepromoter has been altered. In one preferred embodiment, a derivativepromoter will comprise a modification, such as a substitution, of atleast one nucleotide of the −35 box of the promoter.

“Under transcriptional control” or “transcriptionally controlled” areterms well understood in the art that indicate that transcription of apolynucleotide sequence, usually a DNA sequence, depends on its beingoperably linked to an element which contributes to the initiation of, orpromotes, transcription.

“Operably linked” refers to a juxtaposition wherein the elements are inan arrangement allowing them to be functionally related. For example, apromoter is operably linked to a coding sequence if it directstranscription of the sequence.

“Under translational control” is a term well understood in the art thatindicates a regulatory process that occurs after the messenger RNA hasbeen formed.

As used herein the term “gene” means a DNA segment that is involved inproducing a polypeptide and includes regions preceding and following thecoding regions as well as intervening sequences (introns) betweenindividual coding segments (exons).

The terms “polynucleotide” and “nucleic acid”, used interchangeablyherein, refer to a polymeric form of nucleotides of any length, eitherribonucleotides or deoxyribonucleotides. These terms include a single-,double- or triple-stranded DNA, genomic DNA, cDNA, RNA, DNA-RNA hybrid,or a polymer comprising purine and pyrimidine bases, or other natural,chemically, biochemically modified, non-natural or derivatizednucleotide bases. The following are non-limiting examples ofpolynucleotides: a gene or gene fragment, exons, introns, mRNA, tRNA,rRNA, ribozymes, cDNA, recombinant polynucleotides, branchedpolynucleotides, plasmids, vectors, isolated DNA of any sequence,isolated RNA of any sequence, nucleic acid probes, DNA constructs andprimers. A polynucleotide may comprise modified nucleotides, such asmethylated nucleotides and nucleotide analogs, uracyl, other sugars andlinking groups such as fluororibose and thioate, and nucleotidebranches.

A “structural sequence” is a putative polynucleotide sequence of DNAthat encodes the formation of a product. A structural sequence canencode the amino acid sequence of a polypeptide chain having messengerRNA is its primary product. A structural sequence can also encode theformation of an RNA with structural or regulatory function.

As used herein, the term “vector” refers to a polynucleotide constructdesigned to introduce nucleic acids into one or more cell types. Vectorsinclude cloning vectors, expression vectors, shuttle vectors, plasmids,cassettes and the like. In the specific case of a DNA cassette, the DNAmay be generated in vitro by PCR or any other suitable techniques.

The term “over expressed” means an increased number of copies of thesame gene product in a host cell.

The terms “protein” and “polypeptide” are used interchangeabilityherein. The 3-letter code for amino acids as defined in conformity withthe IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN) isused throughout this disclosure. It is also understood that apolypeptide may be coded for by more than one nucleotide sequence due tothe degeneracy of the genetic code.

As used herein when describing proteins, and genes that encode them, theterm for the gene is not capitalized and is italics, i.e. glkA. The termfor the protein is generally not italicized and the first letter iscapitalized, i.e. GlkA.

“A glucose assimilation protein” or “an enzyme involved in glucoseassimilation” means an enzyme or protein that enables a host cell toutilize glucose as a carbon source. Enzymes and proteins involved inglucose assimilation include those involved in glucose transport acrossthe cell membrane and those involved in phosphorylation of glucose toglucose-6-phosphate.

“A phosphorylating enzyme” means an enzyme that catalyzes the reactionof glucose to glucose 6-phospahte.

Enzymes known to catalyze this reaction include hexokinases (E.C. No.:2.7.1.1) and glucokinases (E. C. No. 2.7.1.2). Glucokinase is encoded byglk in E. coli.

A “transport protein” or “transporter” refers to a protein thatcatalyzes the movement of a molecule across a cell membrane. In apreferred embodiment, the transporter, also referred to as a permease isa glucose transporter. A glucose transporter catalyzes the activetransport of glucose across a cell membrane into the cytoplasm. Aglucose transporter may also catalyze the transport of other sugars. Oneexample of a glucose transporter is a galactose-proton symporter (alsoknown as a galactose permease), GalP. GalP is encoded by galP in E. coli(Henderson et al., (1990) Phil. Trans. R. Soc., London 326:391-410).

“Active transport” refers to the transport of a compound into a cellthat is coupled to an expenditure of energy. One example emcompassed bythe invention is the use of membrane potential by glucose transporters.

As used herein a “selectable marker” refers to a gene capable ofexpression in a host cell which allows for ease of selection of thosehosts containing an introduced nucleic acid or vector. Examples of suchselectable markers include but are not limited to antimicrobials (e.g.kanamycin, erythromycin, actinomycin, chloramphenicol, spectinomycin,and tetracycline). The designation “Cm^(R)” and “CAT” for example bothrefer to the same gene, a chloramphenicol resistance gene and also knownas the chloroamphenicol acetyltransferase gene.

A “flanking sequence” refers to any sequence that is either upstream ordownstream of the sequence being discussed (e.g. for genes A, B, and C;gene B is flanked by the A and the C gene sequences). In someembodiments, a flanking sequence is present on only a single side(either 3′ or 5′) of a DNA fragment, but in preferred embodiments, eachside of the sequence being discussed is flanked.

The term wild-type refers to a native or naturally occurring host cellor host cell sequence.

As used herein, the term “endogenous” refers to a nucleic acid orprotein encoded by a nucleic acid naturally occurring in the host. Theterm “exogenous” refers to a nucleic acid or protein from a differenthost cell strain. An exogenous sequence can be a non-host sequence and asynthetically modified native sequence.

The term “homologous” means of the same organism, strain or species. A“homologous sequence” is a sequence that is found in the same geneticsource or species. For example if a host strain lacks a specific gene,but the same gene is found in other strains of the same species the genewould be considered a homologous sequence. The term “heterologous” meansof a different organism, strain or species and more specifically refersto nucleic acid or amino acid sequences not naturally occurring in thehost cell.

The terms “transformation”, “transduction” and “transfection” refer tothe incorporation or introduction of new polynucleotides into a cell.The introduced polynucleotides may be integrated into the chromosomalDNA or introduced as extra chromosomal replicating sequences.

The terms “isolated” or “purified” as used herein refer to an enzyme,nucleic acid, protein, peptide or co-factor that is removed from atleast one component with which it is naturally associated.

“Desired product” as used herein refers to the desired compound intowhich a carbon substrate is bioconverted. Exemplary desired products aresuccinate, lysine, glycerol, methionine, threonine, isoleucine,pyruvate, ethanol, formate, acetate, DAHP, DHQ, DHS, SHK, S3P, EPSP,chorismate, phenylalanine, tyrosine, tryptophan, and ascorbic acidintermediates.

As used herein, the term “carbon source” encompasses suitable carbonsubstrates ordinarily used by microorganisms, such as 6 carbon sugars,including but not limited to glucose (G), gulose, lactose, sorbose,fructose, idose, galactose and mannose all in either D or L form, or acombination of 6 carbon sugars, such as glucose and fructose, and/or 6carbon sugar acids. Preferred carbon substrates include glucose andfructose.

The terms “non-functional”, “inactivated” and “inactivation” whenreferring to a gene or a protein means that the known normal function oractivity of the gene or protein has been eliminated or highlydiminished. Inactivation which renders the gene or proteinnon-functional includes such methods as deletions, mutations,substitutions, interruptions or insertions in the nucleic acid sequence.

A “host cell” is a cell capable of receiving introduced, heterologouspolynucleotides. In one embodiment the host cell is a gram-negative orgram-positive bacteria.

As used herein, the term “bacteria” refers to any group of microscopicorganisms that are prokaryotic, i.e., that lack a membrane-bound nucleusand organelles. All bacteria are surrounded by a lipid membrane thatregulates the flow of materials in and out of the cell. A rigid cellwall completely surrounds the bacterium and lies outside the membrane.There are many different types of bacteria, some of which include, butare not limited to Bacillus, Streptomyces, Pseudomonas, and strainswithin the families of Enterobacteriaceae.

As used herein, the family “Enterobacteriaceae” refers to bacterialstrains having the general characteristics of being gram negative andbeing facultatively anaerobic. Included in the family ofEnterobacteriaceae are Erwinia, Enterobacter, Gluconobacter, Klebsiella,Escherichia, Acetobacter, Coyrnebacteria and Pantoea.

An “altered bacterial host” or “modified bacterial host” according tothe invention is a genetically engineered bacterial cell having aPTS⁻/Glu⁺ phenotype.

An “unaltered bacterial host cell” refers to a bacterial host cell whichuses PTS to transport and phosphorylate glucose or a PTS⁻/Glu⁻ cell.

As used herein “chromosomal integration” is a process whereby anintroduced polynucleotide is incorporated into a host cell chromosome.The process preferably takes place by homologous recombination.Homologous recombination is the exchange of DNA fragments between twoDNA molecules is wherein the homologous sequences of the introducedpolynucleotide align with homologous regions of the host cell chromosomeand the sequence between the homologous regions of the chromosome isreplaced by the sequences of the introduced polynucleotide in a doublecrossover.

A “target site” is intended to mean a predetermined genomic locationwithin a host cell chromosome where integration of a DNA construct is tooccur.

As used herein, “modifying” the level of protein or enzyme activityproduced by a host cell refers to controlling the levels of protein orenzymatic activity produced during culturing, such that the levels areincreased or decreased as desired.

As used herein, the term “modified” when referring to nucleic acid or apolynucleotide means that the nucleic acid has been altered in some wayas compared to a wild type nucleic acid, such as by mutation in;substitution of; insertion of; deletion of part or all of the nucleicacid; or by being operably linked to a transcriptional control region.As used herein the term “mutation” when referring to a nucleic acidrefers to any alteration in a nucleic acid such that the product of thatnucleic acid is partially or totally inactivated. Examples of mutationsinclude but are not limited to point mutations, frame shift mutationsand deletions of part or all of a gene.

A polynucleotide or polypeptide having a certain percentage (forexample, 80%, 85%, 90%, 95%, 96%, 97% or 99%) of “sequence identity” toanother sequence means that, when aligned, that percentage of bases oramino acids are the same in comparing the two sequences. This alignmentand the percent homology or sequence identity can be determined usingsoftware programs known in the art, for example those described inCURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel et al., eds.,1987) Supplement 30, section 7.7.18. Preferred programs include the GCGPileup program, FASTA (Pearson et al. (1988) Proc. Natl. Acad. Sci. USA85:2444-2448) and BLAST (BLAST Manual, Altschul et al., Natl. Cent.Biotechnol. Inf., Natl Library Med. (NCBI NLM), NIH, Bethesda Md. andAltschul et al., (1997) NAR 25:3389-3402). Another preferred alignmentprogram is ALIGN Plus (Scientific and Educational Software,Pennsylvania), preferably using default parameters, which are asfollows: mismatch=2; open gap=0; extend gap=2. Another sequence softwareprogram that could be used is the TFastA Data Searching Programavailable in the Sequence Analysis Software Package Version 6.0 (GeneticComputer Group, University of Wisconsin, Madison, Wis.). One skilled inthe art will recognize that sequences encompassed by the invention arealso defined by the ability to hybridize under stringent conditions withthe sequences exemplified.

A nucleic acid is “hybridizable” to another nucleic acid when a singlestranded form of the nucleic acid can anneal to the other nucleic acidunder appropriate conditions of temperature and solution ionic strength.Hybridization and washing conditions are well known and exemplified inSambrook 1989, supra (see in particular chapters 9 and 11). Lowstringency hybridization conditions correspond to a Tm of 55° C. (forexample, 5×SSC, 0.1% SDS, 0.25 milk and no formamide or 5×SSC, 0.5% SDSand 30% formamide). Moderate stringency conditions correspond to a6×SSC, 0.1% SDS, 0.05% milk with or without formamide, and stringentconditions correspond to for example, a Tm of 65° C. and 0.1×SSC, 0.1%SDS.

It is well understood in the art that the acidic derivatives ofsaccharides and other compounds such as organic acids, may exist in avariety of ionization states depending upon their surrounding media, ifin solution, or out of solution from which they are prepared if in solidform. The use of a term, such as, for example, gluconic acid or aceticacid to designate such molecules is intended to include all ionizationstates of the organic molecule referred to. Thus, for example, “gluconicacid” and “gluconate” refer to the same organic moiety, and are notintended to specify particular ionization states or chemical forms.

The term “culturing” as used herein refers to fermentative bioconversionof a carbon substrate to a desired product within a reactor vessel.Bioconversion means contacting a microorganism with a carbon substrateto convert the carbon substrate to the desired product.

As used herein, the term “comprising” and its cognates are used in theirinclusive sense; that is, equivalent to the term “including” and itscorresponding cognates.

“A,” “an” and “the” include plural references unless the context clearlydictates otherwise.

“Allowing the production of a desired product from a carbon source,wherein the production of the desired product is enhanced compared tothe production of the desired product in a corresponding unalteredbacterial host cell” means contacting the substrate, e.g. carbon source,with the PTS⁻/Glu⁻ bacterial cell to produce the desired product.

PREFERRED EMBODIMENTS

The present invention is directed to a method for increasing carbon flowinto a desired metabolic pathway of a host cell originally capable ofutilizing a PTS for carbohydrate transport, said method including thesteps of selecting a host cell which is effectively phenotypically PTS⁻and modifying at least one homologous chromosomal regulatory region,which is operably linked to a chromosomal nucleic acid which encodes apolypeptide involved in glucose assimilation, resulting in therestoration of a glucose⁺ phenotype and thereby increasing the carbonflow into and through a desired metabolic pathway.

A. PTS Host Cells.

A general review of the PTS can be found in (Postma et al., 1993,Microbiol. Rev. 57:543-594; Romano et al., 1979, J. Bacteriol. 139:93-97and Saier et al. 1990, In: BACTERIAL ENERGETICS pp. 273-299, T. A.Krulwich, Ed. Academic Press, NY). Host cells or strains useful in thepresent invention include any organism capable of utilizing a PTS systemfor carbohydrate transport. This includes prokaryotes belonging to thegenus Escherichia, Corynebacterium, Brevibacterium, Bacillus,Pseudomonas, Streptomyces, Pantoea or Staphylococcus. A list of suitableorganisms is provided in Table 1. The inactivation of the PTS in any ofthese organisms should potentially increase carbon flux and PEP (and PEPprecursor) availability in the cell for alternative metabolic routes andconsequently could increase production of desired compounds (e.g.,aromatics) from such cells.

TABLE 1 Host cell Reference Escherichia coli Postma, et al (1993)Microbiol. Rev. 57: 543-594 Salmonella typhimurium Postma, et al (1993)Microbiol. Rev. 57: 543-594 Klebsiella pneumoniae Postma, et al (1993)Microbiol. Rev. 57: 543-594 Bacillus subtilis Postma, et al (1993)Microbiol. Rev. 57: 543-594 Mycoplasma capricolum Postma, et al (1993)Microbiol. Rev. 57: 543-594 Acholeplasma florum Navas-Castillo et al.(1993) Biochimie 75: 675-679 Staphylococcus aureus Postma, et al (1993)Microbiol. Rev. 57: 543-594 Staphylococcus carnosus Postma, et al (1993)Microbiol. Rev. 57: 543-594 Staphylococcus xylosus Wagner et al. (1993)Mol. Gen. Genet. 24: 33-41 Rhodobacter capsulatus Postma, et al (1993)Microbiol. Rev. 57: 543-594 Rhodopseudomonas sphaeroides Meadow et al.(1990) Annu. Rev. Biochem. 59: 497-542 Streptococcus (Enterococcus)faecalis Postma, et al (1993) Microbiol. Rev. 57: 543-594 Streptococcusmutans Postma, et al (1993) Microbiol. Rev. 57: 543-594 Streptococcussalivarius Postma, et al (1993) Microbiol. Rev. 57: 543-594Streptococcus sanguis Postma, et al (1993) Microbiol. Rev. 57: 543-594Streptococcus sobrinus Chen et al. (1993) Infect. Immun. 61: 2602-2610Erwinia chrysanthemi Postma, et al (1993) Microbiol. Rev. 57: 543-594Xanthmonas campestris Postma, et al (1993) Microbiol. Rev. 57: 543-594Corynebacterium glutamicum Cocaign et al. (1993) Appl. Microbiol. andBiotechnol. 40: 526-530 Brevibacterium lactofermentum K-H Yoon (1993)Abstr. Ann. Meet. Am. Soc. Microbiol. 0-25 Bifidiobacterium breve Degnanet al. (1993) Arch. Microbiol. 160: 144-151 Azospirillum brasilienseChattopadhyay et al. (1993) J. Bacteriol. 175: 3240-3243 Listeriamonocytogenes Mitchell et al., (1993) J. Bacteriol. 175: 2758-2761.Spirocheta aurantia Meadow et al. (1990) Annu. Rev. Biochem. 59: 497-542Lactobacillus brevis Meadow et al. (1990) Annu. Rev. Biochem. 59:497-542 Lactobacillus buchneri Meadow et al. (1990) Annu. Rev. Biochem.59: 497-542 Lactobacillus casei Postma, et al (1993) Microbiol. Rev. 57:543-594 Lactococcus cremoris Benthin et al. (1993) Biotechnol. Bioeng.42: 440-448 Lactococcus lactis Postma, et al (1993) Microbiol Rev. 57:543-594 Pseudomonas aeruginosa Meadow et al. (1990) Annu. Rev. Biochem.59: 497-542 Vibrio alginolyticus Postma, et al (1993) Microbiol. Rev.57: 543-594 Vibrio furnissii Yu et al. (1993), J. Biol. Chem. 268:9405-9409 Vibrio parahaemolytica Meadow et al. (1990) Annu. Rev.Biochem. 59: 497-542

Preferred host strains are those known to be useful in producingaromatic compounds, including strains selected from the generaEscherichia, Corynebacterium, Brevibacterium, Pantoea and Bacillus. Thegenus Pantoea includes all members known to those of skill in the art,including but not limited to P. citrea, P. ananratis, P. stewarti, P.agglomerans, P. punctata and P. terrea. Useful Bacillus strains includecells of B. subtilis, B. licheniformis, B. lentus, B. brevis, B.stearothermophilus, B. alkalophius, B. amyloliquefaciens, B. coaglulans,B. ciculans, B. lautus and B. thuringiensis.

B. Selection of PTS⁻/Glu⁻ Host Cells.

Selection of PTS⁻ cells can be achieved using techniques available tothose skilled in the art. Inactivation will effectively reduce PEPphosphorylation to undetectable levels as measured by PEP-dependentphosphorylation of 2-deoxy-D-glucose using the protocols described byGachelin, G. (1969). Biochem. Biophys. Acta. 34:382-387; Romero, et al.,(1979) J. Bact. 139:93-97; or Bouvet and Grimont., (1987) Ann. Inst.Pasteur/Microbiol. 138:3-13. Also PEP phosphorylation assays are usefulin determining the level of PTS⁻ expression.

PST⁻/Glu⁻ host cells may be selected from PTS wild-type host cells byinactivation of at least one gene encoding part or all of the enzymescomprising the PTS. By way of example, in one embodiment, the PTS isinactivated by the deletion of at least one gene selected from the groupconsisting of ptsI, ptsH and crr encoding the El, HPr and IIA^(Glc)proteins of the PTS respectively (Postma, et al (1993) Microbiol. Rev.57:543-594). In other embodiments, at least two of the genes areinactivated. The nucleotide sequences of ptsI, ptsH and crr have beendetermined (Saffen et al., (1987) J. Biol. Chem. 262:16241-16253; Fox etal., (1984) Biochem. Soc. Trans. 12:155-157; Weigel et al., (1982) J.Biol. Chem. 257:14461-14469 and DeReuse et al., (1988) J. Bacteriol.176:3827-3837). In other embodiments, the inactivation of all threegenes ptsI, ptsH and crr by deletion will effectively reduced PEPphosphorylation to undetectable levels.

Generally, methodology employed in the present invention to inactivatethe PTS is as follows. It is known that in E. coli the ptsI, ptsH andcrr are linked together in an operon. The ptsHIcrr operon in E. colistrains JM101 (Yanisch-Perron et al. (1985) Gene 33:103-119) and strainPB103 (Mascarenhas (1987) PCT WO87/01130) was inactivated by deletionusing a generalized transduction method as described by Silhavy, et al.(1984) In: EXPERIMENTS WITH GENE FUSIONS, pp 110-112, Cold Spring HarborLaboratory Press, NY. P1vir phage was used to perform the transductionand strain TP2811 (Levy et al., (1990) Gene 86:27-33) was used as thedonor of the ptsHIcrr deletion. The process was carried out in twostages. First, a cell-free suspension of phage was prepared by growingbacteriophage P1vir on strain TP2811. In the TP2811 strain most of theptsHIcrr operon has been deleted and a kanamycin-resistant marker wasinserted in the same DNA region (Levy et al., (1990) Gene 86:27-33). Theobtained P1vir lysate was able to transduce the ptsHIcrr deletion andkanamycin resistance marker simultaneously. Secondly, these phage wereused to infect the recipient strains, JM101 or PB103 and transductantswere selected by plating the infected cells on MacConkey-glucose platescontaining kanamycin. After incubating the plates for 16 hours at 37°C., several white colonies appeared.

The recipient strains (JM101 and PB103) are kanamycin sensitive and formred colonies on MacConkey-glucose plates. The MacConkey-glucose platescontain an indicator dye that, depending on the pH, can vary from whiteto deep red. If the cells can transport glucose at a fast rate, normallythey will secrete organic acids and produce red colonies. If glucosetransport is diminished or absent, the cells will not produce organicacids and the colonies will be white. This enables one to ascertainwhether the host cell exhibits a glucose⁺ or glucose⁻ phenotype.

Transduction of the resulting kanamycin resistant colonies were white,which indicates that the ability of the cells to assimilate glucose wasaffected, and it is believed this is due to the transfer of the ptsHIcrroperon deletion. To corroborate this assumption transductants can beselected and inoculated in minimal medium containing glucose as the onlycarbon source. One would expect after incubation (for 12 hours at 37°C.) the transductants would have no detectable cell growth and the PTSparent strains JM101 and P8103 would grow very well and reference ismade to WO 96/34961. This result was observed and based on theseresults, the PTS⁻ derivative of JM101 was designated PB11 and the PTS⁻derivative of P8103 was designated NF6.

Another test for the absence of the PTS system is based on the fact thatPTS⁻ strains become resistant to the antibiotic fosfomycin Cordaro etal., (1976) J. Bacteriol 128:785-793.

While the above methodology is a preferred means of providing aninactivated PTS (PTS⁻/Glu⁻) other methods can also be used. One furthernonlimiting method includes inserting or modifying a repressor bindingregion operably linked with a gene encoding an expressed protein suchthat the expression of the gene occurs in the presence of a specificmolecule. For example, the lac operator is a DNA sequence recognized bythe Lac repressor. If this operator is located in the proper position,relative to the promoter, then if the repressor is bound to theoperator, it will interfere with the binding of the RNA polymerase andblock gene transcription. This blockage can be removed by the additionof the inducer IPTG (isopropyl-β-D-thiogalactoside). The level ofrepression and/or induction will depend on the strength of the promoter,the location and sequence of the operator, as well as the amount of therepressor and the inducer (Muller J. et al., (1996) J. Mol. Biol.257:21-29). The lac operator is used to regulate a number of promoters,among them several variants of the lac promoter and the hybrid trcpromoter.

Another nonlimiting method to affect a PTS⁻/Glu⁻ phenotype includes theincorporation of a promoter which affects the expression of thestructural gene sequence when certain conditions are present. Forexample, the Pm promoter from the TOL plasmid can be used to controlgene expression. In this system, gene expression is achieved whenbenzoate or toluate is added to the media, (Mermod et al., (1986) J.Bact. 167:447-454). Still a further nonlimiting method to affect a PTS⁻phenotype is to alter the mRNA stability as described by Carrier andKeasling (1997) Biotechnol. Prog. 13:699-708.

However, to increase or redirect carbon flow to desired metabolicpathways in inactivated PTS host cells, glucose transport andphosphorylation must be deregulated or amplified.

C. Restoring the Glucose⁺ Phenotype.

While not wanting to be limited by theory, it is thought that themodification of alternative glucose assimilation pathways compensatesfor the inability to actively transport glucose by the PTS, therebyallowing the host cell to utilize PEP otherwise metabolized in thetransport of glucose for other purposes.

Once a PTS⁻/Glu⁻ host cell is obtained, a homologous chromosomalregulatory region operably linked to a chromosomal nucleic acid encodinga polypeptide involved in glucose assimilation is modified to restore aglucose⁺ phenotype, thereby obtaining a PTS⁻/Glu⁺ phenotype. Theregulatory region that is operably linked to the expression of thepolypeptide involved in glucose assimilation can be an operator, apromoter, an inhibitor or a repressor.

In one preferred embodiment, a DNA cassette comprising a regulatoryregion including a promoter is introduced into a PTS⁻/Glu⁻ host cell anda homologous chromosomal regulatory region operably linked to achromosomal nucleic acid encoding a polypeptide involved in glucoseassimilation is modified to restore a glucose⁺ phenotype.

D. Construction of DNA Integration Cassettes Comprising RegulatoryRegions.

Typically a DNA cassette or construct according to the invention whichis useful for modifying endogenous chromosomal regulatory regions in aPTS⁻/Glu⁻ host includes regulatory sequences, such as promoters. Inanother embodiment, the DNA cassette further includes a selectablemarker and sequences allowing autonomous replication or chromosomalintegration, such as recombinase recognition sites. In anotherembodiment the DNA cassette further includes flanking sequences, whichare located upstream (5′) and downstream (3′) of the recombinaserecognition sites.

Promoters—

In one embodiment, the regulatory region comprising the DNA cassetteincludes a promoter. In further embodiments, the promoter is anexogenous promoter. In other embodiments the exogenous promoter is anon-native promoter and derivatives thereof. In further embodiments, thepromoter is a native promoter which in its native endogenous form is notoperably linked to a polynucleotide encoding a glucose assimilationprotein. In some embodiments the exogenous promoter is a modifiednaturally occurring promoter, which in its native endogenous form islinked to a polynucleotide encoding a glucose assimilation protein,wherein the native promoter has been altered and then reintroduced intoa host cell chromosome. For example the native promoter could bemodified at the −35 region, the −10 region or the linker region or thenative promoter could include a modification of a repressor bindingsite. In other embodiments, the native promoter, is one that is notlinked to a polynucleotide encoding a glucose transporter such as agalactose-proton symporter, and more specifically to galP. Further inother embodiments, the native promoter, is one that is not linked to apolynucleotide encoding a phosphorylating protein such as glucokinase,and more specifically is not linked to glk. A regulatory region andspecifically including a promoter useful in a DNA cassette according tothe invention includes sequences of between about 20 to 200 bp, ofbetween about 20 to 150 bp and of between about 20 to 100 bp.

Preferably the promoter will be stronger that the naturally occurringendogenous wild-type promoter and will result in increased expression ofthe glucose assimilation protein. Those skilled in the art willrecognize that various methodologies are known to determine the relativestrength of the promoters. Promoter strength can be quantified using invitro methods that measure the kinetics of binding of the RNA polymeraseto a particular piece of DNA, and also allows the measurement oftranscription initiation (Hawley D. K et al., Chapter 3: in: PROMOTERS:STRUCTURE AND FUNCTION. R. L/Rodriguez and M. J. Chamberlin eds. PraegerScientific. New York). Also in vivo methods may be used also to quantifypromoter strength. For example a promoter can be fused to a reportergene and the efficiency of RNA synthesis measured. Deuschle et al.,(1986) (EMBO J. 5: 2987-2994.) determined the strength of 14 E. colipromoters using 3 different reporter genes. These promoters include thefollowing trc, tac, D/E20, H207, N25, G25, J5, A1, A2, A3, L, Lac,LacUV5, Con, β-lactamase (bla), T5“early” P_(L), and H/McC. Each ofthese promoters or derivatives thereof may be used as exogenouspromoters in accordance with the present invention.

Additionally, a modified naturally occurring promoter and a nativepromoter, which in its native endogenous form is not linked to apolynucleotide encoding a glucose assimilation protein may be usedaccording to the invention. Native promoters may be determined byvarious exemplary methods. While not wanting to be limited, in oneembodiment, sequencing of the particular genome may be performed andputative promoter sequences identified using computerized searchingalgorithms, For example a region of a genome may be sequences andanalyzed for the presence of putative promoters using Neural Network forPromoter Prediction software (NNPP). NNPP is a time delayed neuralnetwork consisting mostly of two feature layers, one for recognizingTATA-boxes and one for recognizing so called, “initiators” which areregions spanning the transcription start site. Both feature layers arecombined into one output unit. The putative sequences may then be clonedinto a cassette suitable for preliminary characterization in E. coliand/or direct characterization in E. coli. In another embodiment,identification of consensus promoter sequences can be identified byhomology analysis, for example by using BLAST. The putative promotersequence may then be cloned into a cassette suitable for preliminarycharacterization in E. coli.

While numerous promoters and their derivatives may be used, preferredpromoters include, the trc promoter and derivatives thereof (Amann etal., (1983) Gene 25:167-178). The trc promoter is illustrated in FIG. 2wherein the −35 box is TTGACA and the −10 box is TATAAT. Anotherpreferred promoter is the tac promoter. The nucleic acid sequence of thetac promoter and the trc promoter is the same with the exception of thelinker region. The linker region of tac promoter differs by 1 bp.(Russell and Bennett (1982) Gene 20:231-243).

Another preferred promoter is a glucose isomerase (GI) promoter (alsoknown as a xylose isomerase promoter). Reference is made to Amore et al.(1989) Appl. Microbiol. Biotechnol. 30:351-357. The sequence of a shortsegment of the GI promoter (+50 to −7 of the −10 box) is set forth inSEQ ID NO. 5 5′CGAGCCGTCACGCCCTTGACAATGCCACATCCTGAGCAAATAAT 3′ whereinthe −35 box is represented by TTGACA and the −10 box is represented byAATAAT.

A derivative promoter may include a modification to at least onenucleotide in a position corresponding to a nucleotide in the −35 box,linker region or −10 box. In a preferred embodiment these derivativepromoters are altered in a position corresponding to a position in the−35 box. Particularly preferred derivative promoters include amodification to a −35 box corresponding to TTGACA and TTTACA. SomeTTGACA modifications include TTGAAA, TTCAC and CTGACA. One particularmodification is to the position corresponding to position −35.Particularly preferred derivative promoters also include a modificationto a −10 box corresponding to TATAAT, TAAGAT and TATGTT. Linker regionsmay also be modified (Burr et al., (2000) NAR 28:1864-1870). Preferablylinker regions whether modified or unmodified are between 14 to 20 bp inlength, preferably 16 bp, 17 bp, 18 bp and 19 bp. Those skilled in theart are well aware of methods used to make modifications in promotersand the present invention is not limited by these methods. One exemplarymethod includes use of the Quikchange Kit (Stratagene); Reference isalso made to WO 98/07846; Russell and Bennett (1982) Gene 231-243 andSommer et al. (2000) Microbiol, 146:2643-2653.

Further preferred derivatives promoters include trc derivativepromoters. The trc derivative promoter may include at least onemodification in the −35 consensus box, the −10 consensus box or thelinker region. The modification may be an insertion, substitution, ordeletion. In a preferred embodiment the trc derivative promoter includesat least one modification to the −35 box. For example in E. coli sincethe codon at position −30 is adenine (A), it may be substituted withthymine (T), guanine (G) and cytosine (C); since the codon at position−31 is C it may be substituted with A, T and G; since the codon atposition −32 is A, it may be substituted with T, G and C; since thecodon at position −33 is G it may be substituted with C, T and A; sincethe codon at position −34 is T it may be substituted with A, C and G;and since the codon at position −35 is T, it may be substituted with A,G and C. One particularly preferred trc derivative promoter includes amodification to the codon at position −35 and most preferably amodification wherein T is substituted with C.

Other preferred trc derivative promoters include a modification in the−10 box. For example, since the nucleotide at −7 is T, it may besubstituted with a nucleotide selected from the group consisting of C,G, and A.; since the nucleotide at −8 is A, it may be substituted with anucleotide selected from the group consisting of C, G, and T; since thenucleotide at −9 is G, it may be substituted with a nucleotide selectedfrom the group consisting of C, T, and A; since the nucleotide at −10 isA, it may be substituted with a nucleotide selected from the groupconsisting of C, G, and T; since −11 is A, it may be substituted with anucleotide selected from the group consisting of T, G, and C, and since−12 is T, it may be substituted with a nucleotide selected from thegroup consisting of A, C, and G.

Selective Markers and Recombinase Recognition Sites—

A DNA cassette encompassed by the invention will include a selectablemarker and a number of genes can be used to detect insertion of the genein E. coli. Some of these genes confer selectable phenotypes. In thiscase, media conditions can be established so that only colonies whichhave expression of these genes activated will grow. Other genes conferphenotypes which can be screened. A screenable phenotype can often yieldinformation about levels of gene expression. While any desired markercan be used, based on these properties, useful antibiotic resistance(Anb^(R)) markers include but are not limited to, Cm^(R), Km^(R) andGm^(R). A preferred non-limiting example of a selectable marker is achloramphenicol acetyltransferase (CAT) gene.

In a preferred embodiment, a DNA cassette comprising the promoter to beintegrated into a host cell chromosome at a target site will include aselectable marker flanked on both sides by a recombinase recognitionsite. Recombinase sites are well known in the art and generally fallinto two distinct families based on their mechanism of catalysis andreference is made to Huang et al., (1991) NAR, 19:443; Datsenko andWarner (2000) Proc. Natl. Acad. Sci. 97:6640-6645 and Nunes-Duby, D, etal (1998) NAR 26:391-406. Preferably the recognition sites are the same.

One well known recombination system is the Saccharomyces Flp/FRTrecombination system, which comprises a Flp enzyme and two asymmetric 34bp FRT minimum recombination sites (Zhu et al., (1995) J. Biol. Chem270:11646-11653). A FRT site comprises two 13 bp sequence inverted andimperfectly repeated, which surround an 8 bp core asymmetric sequencewhere crossing-over occurs. (Huffman et al., (1999) J. Mol. Biol.286:1-13)

One preferred recombinase system is the Cre/loxP site-specificrecombination system of bacteriophage P1, which comprises a Cre enzymeand two asymmetric 34 bp loxP recombination sites (Sternberg andHamilton (1981) J. Mol. Biol. 150:467-486); Palmeros, B, et al. (2000)Gene 247:255-264; Hoess et al. (1986) NAR 14:2287-2300; Sauer B. (1994)Curr. Opinions in Biotechnol. 5:521-527). A loxP site comprises two 13bp sequences, inverted and imperfectly repeated, which surround an 8 bpcore asymmetric sequence, where crossing-over occurs. The Cre-dependentintramolecular recombination between two parallel loxP sites results isexcision of any intervening DNA sequence as a circular molecule,producing two recombination products, each containing one loxP site(Kilby et al., (1993) Trends Genet. 9:414-421).

Homologous Flanking Regions—

An integration DNA cassette according to the invention may also includenucleic acid sequences homologous to upstream (5′) regions of a geneencoding a glucose assimilation protein. These homologous sequences willpreferably flank the first recombinase recognition site (5′ thereto) andthe promoter (3′ thereto). Nucleic acid sequences homologous to upstream(5′) regions of a gene encoding a glucose assimilation protein includesequences derived from a) a sequence 5′ to the endogenous regulatoryregion that is targeted for modification, and b) a sequence 3′ of theendogenous regulatory region that is targeted for modification. The 3′sequence may include parts of a glucose assimilation protein codingsequence. A homologous flanking sequence may include from about 2 to 300bp, about 5 to 200 bp, about 5 to 150 bp, about 5 to 100 bp and about 5to 50 bp.

Isolation of Genes and Glucose Assimilation Proteins—

Methods of obtaining a desired gene from bacterial cells are common andwell known in the art of molecular biology. For example, if a sequenceof a gene is known, suitable genomic libraries may be created andscreened. Once a sequence is isolated the DNA may be amplified usingstandard techniques such as polymerase chain reaction (PCR) (U.S. Pat.No. 4,683,202) to obtain amounts of DNA by restriction. Also referenceis made to Sambrook et al., suprae For the purpose of the presentinvention, upstream sequences as defined above of any gene encoding aglucose assimilation protein is suitable for use in the disclosedmethods.

In one embodiment, a gene encoding a glucose assimilation protein is aglucose transporter. Transporters are discussed in Saier et al., (1998)ADVANCES IN MICROBIAL PHYSIOLOGY, Poole, R. K. Ed. pp 81-136 Academicpress, San Diego, Calif. In general, the glucose transporters as definedherein fail with the Transport Council (TC) classification of transportclass 2 (GALP) and/or transport class 4 (PTS).

A preferred glucose transporter is GalP, which is encoded by galP in E.coli. One of skill in the art will appreciate that genes encoding GalPisolated from sources other than E. coli will also be suitable for usein the present invention. Moreover, proteins functioning as glucosetransporters and having at least 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% and 98% amino acid sequenceidentity to GalP from E. coli will be suitable for use according to theinvention.

Additionally publicly available computer programs can be used todetermine sequences with identity to a glucose assimilation protein andspecifically to a glucose transporter. Preferred programs include theGCG Pileup program, FASTA (Pearson et al. (1988) Proc. Nat. Aced. Sci.USA 85:2444-2448) and BLAST (BLAST Manuael, Altschul et al., Natl. Cent.Biotechnol. Inf., Natl Library Med. (NCBI NLM), NIH, Bethesda Md. andAltschul et al., (1997) NAR 25:3389-3402).

By using BLAST at least 3 permeases with protein sequence similarity toGalP have been found. For example, areE with 64% protein sequenceidentity; xylE with 34% protein sequence identity and yaaU with 23%protein sequence identity. These peremases may also function as glucosetransporters.

In many cases, transporter proteins are highly regulated in cells andcommonly the expression of transporter genes is induced by the presenceof a substrate in the media. The glucose transporter, galactose permease(GalP) from E. coli is induced by galactose, but the preferred substrateis glucose (Henderson & Maidenn (1990). Phil. Trans. R. Soc. Lond. 326:391-410). In E. coli, besides GalP, other permeases can recognize andtransport glucose, for example:

-   -   (a) the high affinity galactose transport system encoded by the        mglBCA genes (Hogg et al. (1991) Mol. Gen. Genet. 229:453-459        and Ferenci T. (1996) FEMS Microbiol. Rev. 18:301-317); and    -   (b) the mannose PTS system, where the membrane component PtsM        has a broad substrate specificity and is capable of transporting        glucose and fructose (Postma & Lengeler (1985) Microbial Rev.        49:232-269 and Erni et al., (1987) J. Biol. Chem.        262:5238-5247).        Further the product of the ptsG gene, which normally is part of        the glucose-PTS system, can be converted by mutagenesis to a        PTS-independent transporter, that functions as a glucose        assimilation protein and more specifically as a        glucose-facilitator. (Ruijter et al (1992) J. Bact. 174:        2843-2850 and Erni et al (1986) J. Biol. Chem. 261:16398-16403).

Besides the well characterized examples above, other glucosetransporters include those cataloged in the TransportDB database. Thisis a relational database describing the predicted cytoplasmic membranetransport protein complement for organisms whose complete genomesequence is available (http://66.93.129.133/transporter/wb/index.html).

In another embodiment, the glucose assimilation protein is aphosphorylating protein. The phosphorylating protein may be a hexokinaseand preferably is a glucokinase. One preferred glucokinase is Glk andreference is made to NCBI (NC 000913). As indicated above for glucosetransporters, other glucose phosphorylating enzymes may be identifiedusing the computer programs such as FASTA, GCG Pileup and BLASTA.

E. coli includes other glucose phosphorylating enzymes as suggested bythe result of Flores et al. (2002) Met. Eng. 4:124-137 and Curtis et al.(1975) J. Bacteriol, 122:1189-1199). When glk was interrupted in E.coli, the cells had a residual glucose phosphorylating activity of 22 to32% compared to a wild-type strain. A BLAST search of the E. coli genomeusing the Glk sequence, did not showed any protein with a level ofsequence identity of greater than 34%. This may indicate that themeasured glucokinase activity depends on one or more enzyme(s) not ordistantly related to Glk. Some of these glucose assimilation proteinswhich may contribute to glucokinase activity are listed below:

Gene name Current annotation in the NCBI database gntK gluconate kinase2 NP 417894 idnK gluconate kinase NP 418689 kdgK KetodeoxygluconokinaseNP 417983 galK Galactokinase NP 415278 pfkA 6-phosphofructokinase NP418351 rbsK ribokinase NP 418208 fruK 1-phosphofructokinase NP 416673yoaC(b1511) putative kinase NP 416028 yajF(b0394) possibletranscriptional regulator NP 414928 ycfX(b1119) putative transcriptionalregulator NP 415637 fucl L-fuculokinase NP 417283

This list is not exhaustive, and it is suggested by the inventors thatby using the proper mutagenesis-selection protocols, these and/or otherkinases can be modified to increase their ability to phosphorylateglucose.

E. Introduction of DNA Cassettes into PTS⁻/Glu⁻ Cells—

Once suitable DNA cassettes are constructed they may be introduced intoplasmids or directly used to transform appropriate PTS⁻/Glu⁻ host cells.Plasmids which can be used as vectors in bacterial organisms are wellknown and reference is made to Maniatis, et al., MOLECULAR CLONING: ALABORATORY MANUAL, 2d Edition (1989) AND MOLECULAR CLONING: A LABORATORYMANUAL, second edition (Sambrook et al., 1989) and Bron, S, Chapter 3,Plasmids, in MOLECULAR BIOLOGY METHODS FOR BACILLUS, Ed. Harwood andCutting, (1990) John Wiley & Sons Ltd.

Useful vectors in the present invention include the vectors pSYCO101(FIGS. 8 and 9), and the pSYCO 101 derivative plasmids pSYCO103 andpSYCO106; pKD46; pR6K-ECHO (Invitrogen); pJW168 (Palmeros et al., 2000Gene, 247:255-264); ptrcM2; ptrc99A; pTrc99 (Pharmacia); pACYC177;pMCGG; pSC101; pKD46 (Datsenko and Wanner (2000) PNAS 97:6640-6645); andpKP32 WO 01/012833).

Introduction of a DNA cassette and other vectors into a host cell may beaccomplished by known transfer techniques. These gene transfertechniques include transformation, transduction, conjugation andprotoplast fusion. Gene transfer is the process of transferring a geneor polynucleotide to a cell or cells wherein exogenously added DNA istaken up by a bacterium. General transformation procedures are taught inCURRENT PROTOCOLS IN MOLECULAR BIOLOGY (vol. 1, edited by Ausubel etal., John Wiley & Sons, Inc. 1987, Chapter 9) and include calciumphosphate methods, transformation using DEAE-Dextran andelectroporation. A variety of transformation procedures are known bythose of skill in the art for introducing nucleic acids in a given hostcell. (Reference is also made to U.S. Pat. No. 5,032,514; Potter H.(1988) Anal, Biochem 174:361-373; Sambrook, J. et al., MOLECULARCLONING: A LABORATORY MANUAL, Cold Spring Harbor Laboratory Press(1989); and Ferrari et al., Genetics, pgs 57-72 in BACILLUS, Harwood etal., Eds. Plenum Publishing Corp).

The introduction of a DNA cassette comprising a promoter and upstreamsequences of a gene encoding a glucose assimilation protein into aPTS⁻/Glu⁻ host cell results in modification of the endogenouschromosomal regulatory region, preferably by replacement of theendogenous regulatory region. In one embodiment, the DNA cassetteincludes an exogenous promoter and the promoter of the endogenousregulatory region is replaced. The introduced regulatory sequence(including the promoter) is chromosomally integrated into the PTS⁻/Glu⁻cell and the introduced sequence becomes operably linked with a codingsequence of the glucose assimilation protein replacing endogenousregulatory sequences. In a preferred embodiment, a selectable markergene, which was introduced with the integrating DNA cassette is removed,preferably by using the methods described in Palmeros et al. (2000) Gene247:255-264. The expression of the glucose assimilation protein, whichis linked to the exogenous promoter, results in a cell having a glucose⁺phenotype. Bacterial strains having a glucose⁺ phenotype (PTS⁻/Glu⁺), asdisclosed herein are also encompassed by the present invention.

In a further embodiment, the modified endogenous regulatory region isthe regulatory region of a glucose transporter. In a preferredembodiment, the glucose transporter gene encodes GalP from E. coli andglucose transporters having at least 60%, 70%, 75%, 80%, 85%, 90%, 95%,97% and 98% amino acid sequence identity to GalP from E. coli. Inanother embodiment, the modified endogenous regulatory region is theregulatory region of a phosphorylating protein. In a preferredembodiment, the phosphorylating protein is a glucokinase and morepreferrably a Glk from E. coli and phosphorylating proteins having atleast 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97% and 98% amino acid sequenceidentity to Glk from E. coli.

In one embodiment, the obtained PTS⁻/Glu⁺ cells having a modifiedendogenous regulatory sequence operably linked to a glucose assimilationprotein, and particularly to a modified endogenous regulatory sequenceoperably linked to a galactose permease and a modified endogenousregulatory sequence operably linked to a glucokinase according to theinvention, will be capable of increased production of a desired compoundcompared to a wild type host cell. The desired compounds include thecompounds illustrated in FIG. 1B. Particularly preferred compoundsinclude dihydroxyacetone-P, glycerol, 1,3-propanediol, pyruvate,lactate, chorismate, tryptophan, phenylalanine, tyrosine, oxaloacetate,aspartate, asparagine, tyrosine, succinate, ethanol, and acetyl-CoA. Inparticular the desired compounds include pyruvate, chorismate andsuccinate.

F. Further Modifications of PTS⁻/Glu⁺ Cells—

The PTS⁻/Glu⁺ cells obtained according to the method of the inventionmay further include heterologous polynucleotides encoding one or moreproteins which direct carbon flow into and through the common aromaticpathway. The heterologous polynucleotide may be introduced into aPTS⁻/Glu⁺ cell either prior to, during or following the reversion to aGlu⁺ phenotype.

In one embodiment a PTS⁻/Glu⁺ cell according to the invention mayoverexpress a transketolase, which is encoded by a tktA or tktB.Transketolase is a pentose phosphate pathway enzyme that catalyzes twoseparate reactions each of which produces E4P as a product. (See FIG.1). Amplification (overexpression) of the tktA gene, by introduction ofnucleic acid sequences encoding transketolase may result in an increasein intracellular concentrations of the aromatic precursor E4P (U.S. Pat.No. 5,168,056).

In another embodiment, one or more of the genes (aroG, aroF and aroH)encoding DAHP synthase may be introduced or amplified in a PTS⁻/Glu⁺cell according to the invention. The increased expression of both E4Pand DAHP synthase can result in a significant increase in carboncommitted to the aromatic pathway compared to strains containingelevated DAHP synthase activity alone (U.S. Pat. No. 5,168,056).

Thus in one embodiment the invention concerns a host cell which createsa surge of carbon flow due to the amplification of transketolase inaddition to a host cell which conserves PEP via inactivation of the PTS(PTS⁻).

It should be noted that as the host cell is cultured in conditions whichcreate an increase in carbon flow into the aromatic pathway, it may benecessary to identify and overcome rate-limiting steps in the pathway.This methodology is available to the artisan, see, for example, U.S.Pat. Nos. 5,168,056 and 5,776,736.

As an example, in the following conversion

under conditions that create a surge of carbon flow into the pathway of,for example PTS⁻/Glu⁺ and Tkt overexpressed strains, the activity levelof DHQ synthase is insufficient to consume DAHP as fast as it is formed.As a result of this natural rate-limiting step at aroB, DAHP accumulatesand is excreted into the culture supernatant. This allows DAHPaccumulation to be used as a means of testing the increasedintracellular PEP levels resulting from the PTS⁻/Glu⁺ strains.

In addition to increasing the carbon flux through the aromatic pathway,the following genes may be overexpressed in PTS⁻/Glu⁺ cells according tothe invention: pps which encodes PEP synthase in E. coli (see U.S. Pat.No. 5,985,617) and talA which encodes transaldolase (lida et al. (1993)J. Bacterial. 175:5375-5383). Further any gene encoding an enzyme thatcatalyzes reactions within the common aromatic pathway (for example,DAHP synthase (aroF, aroG, aroH), DHQ synthase (aroB), DHQ dehydratase(aroD), shikimate dehydrogenase (aroE), shikimate kinase (aroL, aroK),EPSP synthase (aroA) and chorismate synthase (aroC) may be amplified inthe PTS⁻/Glu⁺ cells encompassed by the present invention.

It will be readily apparent to those skilled in the art, that a varietyof different genes can be overexpressed depending on the desiredproduct.

In one embodiment, if the desired product is chorismate, a PTS⁻/Glu⁺cell according to the invention may overexpress any one of the genes inthe aromatic pathway including the genes coding for the enzymes DAHPsynthase, DHQ synthase; DHQ dehydratase; shikimate dehydrogenase;shikimate kinase; EPSP synthase and chorismate synthase.

In one embodiment, if the desired product is tryptophan, any of thegenes in the tryptophan-specific segment of the aromatic pathway may beamplified, including the genes coding for the enzymes tryptophansynthase (trpA and trpB), phosphoribosyl anthranilateisomerase-indoleglycerol phosphate synthase (trpC), anthranilatephosphoribosyl transferase (trpD) and anthranilate synthase (trpE). Inanother embodiment the gene (tnaA) encoding tryptophanase may bedeleted.

In another embodiment, if the desired product is pyruvate a PTS⁻/Glu⁺cell according to the invention may be genetically engineered tooverexpress pyk. This gene encodes a pyruvate kinase. If the desiredcompound is oxaloacetate a PTS⁻/Glu⁺ cell according to the invention maybe genetically engineered to overexpress a ppc which encodes a PEPcarboxylase (EC 4.1.1.31).

If the desired compound is catechol, the PTS⁻/Glu⁺ cell according to theinvention may be further transformed with DNA encoding one or more ofthe following enzyme(s): DAHP synthase (aroF, aroG, aroH);3-dehydroquinate (DHQ) synthase (aroB); transketolase (tktA or tktB);3-dehydroshikimate (DHS) dehydratase (aroZ) or protocatechuate (PCA)decarboxylase (aroY) (see U.S. Pat. Nos. 5,272,073 and 5,629,181).

Furthermore, by way of example, if the desired product is adipic acid,one or more of the following enzyme(s) may be overexpressed (byamplification of the corresponding gene): 3-dehydroshikimate (DHS)dehydratase (aroZ); protocatechuate (PCA) decarboxylase (aroY) orcatechol 1,2-dioxygenase (catA); and, optionally, transketolase (tktA ortktB); DAHP synthase (aroF, aroG, aroH) or DHQ synthase (aroB) in aPTS⁻/Glu⁺ cell according to the invention. (See U.S. Pat. No.5,487,987).

If the desired product is indigo, the PTS⁻/Glu⁺ cell according to theinvention may be further transformed with DNA encoding a polypeptideanalog of a tryptophan synthase beta-subunit and DNA encoding anaromatic dioxygenase enzyme. (See U.S. Pat. No. 5,374,543).

Thus, having provided a PTS⁻/Glu⁺ strain which conserves PEP resultingin an increase in carbon flux into a metabolic pathway, such as thearomatic amino acid pathway, glycolysis, the TCA cycle, and the pentosephosphate pathway, by redirecting PEP and PEP precursors, the inventorshave provided a host system which can be utilized for enhancedproduction of desired compounds in comparison to the production of thesame compounds in a corresponding PTS host cell.

G. Cell Cultures and Fermentations—

Methods suitable for the maintenance and growth of bacterial cells arewell known and reference is made to the MANUAL OF METHODS OF GENERALBACTERIOLOGY, Eds. P. Gerhardt et al., American Society forMicrobiology, Washington D.C. (1981) and T. D. Brock in BIOTECHNOLOGY: ATEXTBOOK OF INDUSTRIAL MICROBIOLOGY, 2nd ed. (1989) Sinauer Associates,Sunderland, Mass.

Cell Precultures

Typically cell cultures are grown at 25 to 32° C., and preferably about28 or 29° C. in appropriate media. Exemplary growth media useful in thepresent invention are common commercially prepared media such as but notlimited to Luria Bertani (LB) broth, Sabouraud Dextrose (SD) broth orYeast medium (YM) broth. These may be obtained from for example,GIBCO/BRL (Gaithersburg, Md.). Other defined or synthetic growth mediamay be used and the appropriate medium for growth of the particularbacterial microorganism will be known by one skilled in the art ofmicrobiology or fermentation science. Suitable pH ranges preferred forthe fermentation are between pH 5 to pH 8. Preferred ranges for seedflasks are pH 7 to pH 7.5 and preferred ranges for the reactor vesselsare pH 5 to pH 6. It will be appreciated by one of skill in the art offermentation microbiology that a number of factors affecting thefermentation processes may have to be optimized and controlled in orderto maximize the ascorbic acid intermediate production. Many of thesefactors such as pH, carbon source concentration, and dissolved oxygenlevels may affect enzymatic processes depending on the cell types usedfor ascorbic acid intermediate production.

The production of desired products can proceed in a fermentativeenvironment, that is, in an in vivo environment, or a non-fermentativeenvironment, that is, in an in vitro environment; or combined in vivo/invitro environments. The fermentation or bioreactor may be performed in abatch process or in a continuous process.

Fermentation Media

Fermentation media in the present invention must contain suitable carbonsubstrates which will include but are not limited to monosaccharidessuch as glucose, oligosaccharides such as lactose or sucrose,polysaccharides such as starch or cellulose and unpurified mixtures froma renewable feedstocks such as cheese whey permeate, cornsteep liquor,sugar beet molasses, and barley malt. Additionally the carbon substratemay also be one-carbon substrates such as carbon. While it iscontemplated that the source of carbon utilized in the present inventionmay encompass a wide variety of carbon containing substrates and willonly be limited by the choice of organism, the preferred carbonsubstrates include glucose and/or fructose and mixtures thereof. Byusing mixtures of glucose and fructose in combination with the modifiedgenomes described elsewhere in this application, uncoupling of theoxidative pathways from the catabolic pathways allows the use of glucosefor improved yield and conversion to the desired ascorbic acidintermediate while utilizing the fructose to satisfy the metabolicrequirements of the host cells.

Although it is contemplated that all of the above mentioned carbonsubstrates are suitable in the present invention preferred are thecarbohydrates glucose, fructose or sucrose. The concentration of thecarbon substrate is from about 55% to about 75% on a weight/weightbasis. Preferably, the concentration is from about 60 to about 70% on aweight/weight basis. The inventors most preferably used 60% or 67%glucose.

In addition to an appropriate carbon source, fermentation media mustcontain suitable minerals, salts, vitamins, cofactors and bufferssuitable for the growth or the cultures and promotion of the enzymaticpathway necessary for ascorbic acid intermediate production.

Batch and Continuous Fermentations

The present process employs either a batch, fed-batch or continuousfermentation method for its culture systems. These methods are wellknown in the art and examples may be found in Brock, supra. A classicalbatch fermentation is a closed system where the composition of the mediais set at the beginning of the fermentation and not subject toartificial alterations during the fermentation. Thus, at the beginningof the fermentation the media is inoculated with the desired organism ororganisms and fermentation is permitted to occur adding nothing to thesystem. Typically, however, a “batch” fermentation is batch with respectto the addition of carbon source and attempts are often made atcontrolling factors such as pH and oxygen concentration. In batchsystems the metabolite and biomass compositions of the system changeconstantly up to the time the fermentation is stopped. Within batchcultures cells moderate through a static lag phase to a high growth logphase and finally to a stationary phase where growth rate is diminishedor halted. If untreated, cells in the stationary phase will eventuallydie. Cells in log phase generally are responsible for the bulk ofproduction of end product or intermediate.

A variation on the standard batch system is the Fed-Batch system.Fed-Batch fermentation processes are also suitable in the presentinvention and comprise a typical batch system with the exception thatthe substrate is added in increments as the fermentation progresses.Fed-Batch systems are useful when catabolite repression is apt toinhibit the metabolism of the cells and where it is desirable to havelimited amounts of substrate in the media. Measurement of the actualsubstrate concentration in Fed-Batch systems is difficult and istherefore estimated on the basis of the changes of measurable factorssuch as pH, dissolved oxygen and the partial pressure of waste gasessuch as CO₂.

Although in the present invention a batch or fed-batch method ispreferred, a continuous fermentation method may also be used. Continuousfermentation is an open system where a defined fermentation media isadded continuously to a bioreactor and an equal amount of conditionedmedia is removed simultaneously for processing. Continuous fermentationgenerally maintains the cultures at a constant high density where cellsare primarily in log phase growth. Continuous fermentation allows forthe modulation of one factor or any number of factors that affect cellgrowth or end product concentration. For example, one method willmaintain a limiting nutrient such as the carbon source or nitrogen levelat a fixed rate and allow all other parameters to moderate. In othersystems a number of factors affecting growth can be altered continuouslywhile the cell concentration, measured by media turbidity, is keptconstant. Continuous systems strive to maintain steady state growthconditions and thus the cell loss due to media being drawn off must bebalanced against the cell growth rate in the fermentation. Methods ofmodulating nutrients and growth factors for continuous fermentationprocesses as well as techniques for maximizing the rate of productformation are well known in the art of industrial microbiology and avariety of methods are detailed by Brock, supra.

The manner and method of carrying out the present invention may be morefully understood by those of skill in the art by reference to thefollowing examples, which examples are not intended in any manner tolimit the scope of the present invention or of the claims directedthereto. All references and patent publications referred to herein arehereby incorporated by reference.

EXPERIMENTAL Example 1 Construction of PTS⁻ E. coli Strains with trcPromoters

A). Construction of the loxP-CAT-loxP-trc Cassette, Plasmid pTrcM42.

Linear DNA was obtained from plasmid pTrc99a (Pharmacia) digested withthe restriction enzymes HindIII and NcoI according to the suppliersinstructions (New England Biolabs). After purification, the ends of thedigested DNA were filled by T4DNA polymerase as described by Sambrook etal. supra. The resulting blunt-end, linear DNA was circularizedaccording standard protocols and transformed into E. coli TOP-10competent cells (Invitrogen, Carlsbad, Calif.). Cells were plated onLuria-agar (LA) plates (LB medium containing 5 g/L yeast extract; 10 g/Ltryptone, and 10 g/L NaCl plus 2% agar) containing 50 micrograms/ml ofcarbenicillin. After 16 hrs. of incubation at 37° C., four colonies werechosen for further analysis. Purified plasmid DNA was obtained fromthese colonies and subjected to restriction enzymes analysis. It wasconfirmed that the 4 colonies contained the same plasmid and that theDNA region between HindIII and NcoI was deleted. The resulting plasmidwas named pTrc1.

Plasmid pTrc1 contains only one recognition site for the restrictionenzyme BspM1, located approximately 120 bp upstream of the −35 region ofthe trc promoter. That location was selected to introduce an excisableselectable marker. pTrc1 was digested with the BspM1 enzyme according tothe instructions of the supplier (New England Biolabs). The linear pTrc1was gel-purified using a QIAquick gel extraction kit (QIAGEN), filled inwith T4 DNA polymerase as described by Sambrook, supra, and ligated to aloxP-CAT-loxP DNA cassette. The loxP-CAT-loxP DNA cassette was obtainedfrom plasmid pLoxCAT2 (Palmeros et al., (2000) Gene 247:255-264)digested with Stu1 and Bam H1. The ligation mixture was transformed intoE. coli TOP-10 competent cells (Invitrogen) and plated on Luria-agarplates containing 50 micrograms/ml of carbenicillin and 20 micrograms/mlof chloramphenicol. After 16 hrs of incubation at 37° C., severalcolonies appeared on the plate. Some of these colonies were transferredto a fresh LB plate containing carbenicillin and chloramphenicol. Afterplasmid purification and restriction enzyme analysis, two plasmidscontaining the loxP-CAT-loxP-trc with the loxP-CAT-loxP cassette in thesame and in the opposite orientation relative to the trc promoter wereselected and designated pTrcm41 and pTrcm42 (FIG. 6).

B). Construction of a trc Promoter Replacement Template for galP(pR6KgalP)

A DNA cassette containing the trc promoter and lac operator with anupstream loxP-CAT-loxP cassette was amplified by PCR from pTrcm42 usingthe primer set of GalA/GalP2

GalA: (SEQ ID NO. 6) 5′ TCGGTTTTCACAGTTGTTACATTTCTTTTCAGTAAAGTCTGGATGCATATGGCGGCCGCAT 3′ GaLP2: (SEQ ID NO. 7)5′ CATGATGCCCTCCAATATGGTTATTTTTTATTGTGAATTAGTCTGTT TCCTGTGTGAAATTGTTA.3′The primer pair incorporated 40 bp of homology to the galP upstreamregion to each end of the PCR product. The amplification used 30 cyclesof (95° C. for 1 min; 55° C. for 1 min; 72° C. for 2 min) using Taqpolymerase (Roche). This DNA cassette was cloned into Echo pUni/His5 R6Kvector (Invitrogen) and transformed into E. coli Pir1 cells. Positiveclones were confirmed by restriction enzyme digest to release thefragment. This construct was designated pR6KgalP.C). Construction of a trc Promoter Replacement Template for glk(pR6Kglk).

A DNA cassette containing the trc promoter and lac operator with anupstream loxP-CAT-loxP cassette was amplified by PCR from pTrcm42 usingthe primer set of GlkA/Glk2.

GlkA: (SEQ ID NO. 8) 5′-ACTTAGTTTGCCCAGCTTGCAAAAGGCATCGCTGCAATTGGATGCATATGGCGGCCGGAT 3′ Glk2: (SEQ ID NO. 9)5′-CATTCTTCAACTGCTCCGCTAAAGTCAAAATAATTCTTTCTCGTCTG TTTCCTGTGTGAAATTGTTA3′The primer pair incorporated 40 bp of homology to the glk upstreamregion to each end of the PCR product. The amplification used 30 cyclesof (95° C. for 1 min; 55° C. for 1 min; 72° C. for 2 min) using Taqpolymerase (Roche). This DNA cassette was cloned into Echo pUni/His5 R6Kvector (Invitrogen) and transformed into E. coli Pir1 cells. Positiveclones were confirmed by restriction enzymes and the plasmid wasdesignated pR6KglkD). Construction of an E. coli ptsHIcrr Deletion Strain KLpts7

A PTS⁻ (Δ ptsHIcrr) strain of E. coli KLndh81 (KLp23ndh), was obtainedby replacing the entire operon comprising ptsH, ptst and crr with akanamycin resistance marker (Levy et al., (1990) Gene 86:27-33). Thiswas done by P1vir transduction using the phage lysate 2611 NF9pykF::Gmas described in Flores et al., (1996) Nature Biotechnol., 14:620-623.The deletion of the operon was confirmed by amplification of the regionby PCR with primers (ptsHF/crrR) that hybridized to regions upstream ordownstream of the deletion, and by plating the colonies on MacConkey(lactose⁻) agar+1% glucose.

ptsHF (SEQ ID NO. 23) 5′ AGAATTGCAACAGTAATGCCAGCTTGTTAAAAATGCGTA 3′ crrR(SEQ ID NO.24) 5′ CCTGTTTTGTGCTCAGCTCATCAGTGGCTTGCTGAA 3′Those colonies with a deletion in the glucose::PTS system exhibited awhite phenotype on these plates as they were no longer able to utilizethe glucose and as generate acid. This strain was designated KLpts7.E). Replacement of the Natural Promoter of galP with the SyntheticExogenous trc Promoter by Linear DNA Cassette Transformation.

A DNA cassette comprising a loxP-CAT-loxP-ptrc with 40 bp of flankingDNA on each end with homology to the E. coli galP upstream region wasgenerated using rTth RNA polymerase (Perkin Elmer), pR6K-galP (SEQ IDNO. 1) as the template and the primer pairs GalA/GalP2 (SEQ ID NO. 6/SEQID NO. 7). The PCR product was transformed into electro-competent KLpts7cells containing pKD46 for integration using the lambda Red system asdescribed in Datsenko and Wanner (2000), Proc. Natl. Acad. Sci. USA97:6640-6645. Integration of this cassette resulted in the replacementof the regulatory region from 36-183 bp upstream of the galP ATG startcodon. (For reference see GenBank Accession # U28377) with aloxP-CAT-loxP-ptrc cassette to provide strain KLpts::galP-trc.

Colonies were selected on LA plates containing 10 μg/ml chloramphenicol.The integration was confirmed by PCR analysis using the primer pairGalA/GalP2 (SEQ ID NO.6/SEQ ID NO. 7) (amplifying the integration siteto give a 1.4 kb product) and the primer pair GalB1/GalC11 (amplifyingthe integration site, including upstream and downstream regions to givea 2.1 kb product).

GalB1 (SEQ ID NO. 10) 5′ ACTTTGGTCGTGAACATTTCCCGTGGGAAA 3′ GalC11 (SEQID NO. 11) 5′ AGAAAGATAAGCACCGAGGATCCCGATA 3′PCR parameters were 1 min at 95° C.; 1 min at 55° C.; 2 min at 72° C.,30 cycles using Taq DNA polymerase or rtth polymerase as suggested bythe manufacturer. This strain, KLpts::galP-trc, was plated on MacConkeyagar (lactose⁻)+1% glucose. The colonies exhibited a slightly redphenotype compared to KLpts7 which was white, indicating that the formerstrain was able to make acid from glucose while the latter strain(parent) was not. This confirmed the expression of galP and that theGalP allowed uptake of glucose. The promoter region was sequenced toconfirm the presence of the promoter. The chloramphenicol marker wasremoved using the Cre recombinase as described in (Palmeros et al.,(2000) Gene 247:255-264 and the removal was confirmed by PCR using theprimer set GalB1/GalC11 (SEQ ID NO. 10/SEQ ID NO. 11). The resultantstrain was designated KLgalP-ptrc.F). Replacement of the Natural Glucokinase Promoter with the SyntheticExogenous trc Promoter (KLGG) by Linear DNA Cassette Transformation.

A DNA cassette consisting of loxP-CAT-loxP-ptrc with 40 bp of flankingDNA with homology to the upstream region of glk was prepared asdescribed for the galP DNA cassette in example 1E) above. The primer setused was GlkA/Glk2 (SEQ ID NO. 8 and SEQ ID NO. 9) that adds theflanking DNA from 149-189 upstream of the glk ATG to the 5′ end and fromglk ATG to 37 bp upstream of the ATG to the 3′ end (glk accession numberAE000327). The template used for the PCR amplification was pR6Kglk withthe rTth polymerase (Perkin Elmer). The glk-trc DNA cassette wastransformed into electro-competent KLgalP-trc cells containing the pKD46plasmid as described by Datsenko and Wanner (2000) supra. Positiveclones were selected on LA agar containing 10 μg/ml chloramphenicol.Integration of the cassette was confirmed by PCR using the primer setGlkB1/GlkC11 and the PCR program described in construction ofKLgalP-trc. GlkB1 is the forward primer that binds beginning at 700 bpupstream of the glk ATG and GlkC11 binds beginning at 500 bp downstreamof the glk ATG start codon.

GlkB1 (SEQ ID NO. 12) 5′ AACAGGAGTGCCAAACAGTGCGCCGA 3′ GlkC11 (SEQ IDNO. 13) 5′ CTATTCGGCGCAAAATCAACGTGACCGCCT 3′

Colonies were plated onto MacConkey agar (lactose-)+1% glucose. Coloniesexhibited a deep red color, indicating an increase in the conversion ofglucose to acid compared to the parent (KLgalP-trc). The chloramphenicolmarker was removed using the Cre recombinase as described in Palmeros etal., supra and removal was confirmed by PCR using the primer setGlkB1/GlkC11 to give a 1.3 kb product. The resultant strain wasdesignated KLGG.

G). Construction of the PEP-Independent Glucose Transport System fromptrc Cloned into pACYC177 (pMCGG).

A moderate copy number plasmid that allowed expression of galP and glkfrom the trc promoter was constructed. A 3040 bp Accl fragmentcontaining galP and glk each under the control of trc promoters wasisolated from plasmid pVHGalPglk11 (FIG. 13). Plasmid pVHGalPglk11 is alow copy number plasmid derived from the pCL1920 vector (Lerner et al.(1990) NAR 18:4631) that carries the resistance to the Spectinomycinantibiotic and the galP and glk genes from E. coli under the control oftrc promoters. The nucleotide sequence (SEQ ID NO. 25) of the 3040 bpDNA fragment obtained by AccI digestion is illustrated in FIG. 14A-E.The ends were filled in using standard procedures (Sambrook et al.,supra), This blunt-ended fragment was cloned into the ClaI site ofpACYC177 (New England Biolabs) thereby inactivating the kanamycinresistance gene. Colonies were screened for growth on carbenicillin (100micrograms/ml) and lack of growth on kanamycin (10 micrograms/ml).Plasmid DNA was isolated from a positive clone by standard method andthe presence of the desired fragment was confirmed by restriction enzymedigestion using XbaI which cuts only 1 time within the cloned fragment,and separately with BamHI which has two recognition sites in theplasmid. This enabled the inventors to determine the orientation of theinserted fragment. This plasmid was designated pMCGG.

H). Construction of the pSYCO Constructs.

The utility of the PTS⁻/Glu⁺ strains (examples 1E-G) to convert carbonfrom glucose to a product was tested by plasmids carrying genes encodingenzymes that carry out conversion of DHAP to 1, 3 propanediol. The pSYCOconstructs were pSC101 (Stratagene) based plasmids that carry genes forconversion of DHAP (dihydroxyacetone-P) to glycerol (dar1 and gpp2) fromSaccharomyces cerevisiae (referred to as the glycerol pathway) andsubsequently glycerol to 1,3-propanediol (dhaB1-3, dhaX, orfW, X, and Yfrom Klebsiella, (referred to as the 1,3-propanediol pathway). The pSYCOconstructs used in the current examples were pSYCO101, 103 and 106 andreference is made to FIGS. 8 and 9 which depict the nucleotide sequenceand plasmid map of pSYCO 101, respectively. The pSYCO103 construct isidentical to pSYCO101 except the DNA region which includes the glycerolpathway genes and the two EcoR1 sites in the opposite orientation tothat of pSYCO101. The pSYCO106 construct is identical to pSYCO103 exceptfor the removal of the 126 bp of non-coding plasmid DNA between theEcoR1 sites and bp 10589-11145 as indicated in FIG. 9. For theexperiments described herein, the plasmids are functionally equivalent.

Example 2 Constitutive Expression of galP Encoding Galactose Permeasefrom the Chromosome of a Strain Lacking a PEP-PTS System for GlucoseUptake

The production of glycerol and 1,3-propanediol in an PTS⁻/Glu⁺ E. colistrain having a Glu+ phenotype was determined. The PTS⁻/Glu⁺ E. colistrain was obtained by transformation of the PTS⁻/Glu⁻ strain (KLpts7)with pMCGG (example 1G) by standard procedures (Sambrook et al. supra)to create KLpts7/pMCGG or by chromosomal integration of the trc promoterto replace the endogenous native galP promoter in KLpts7 creating aKlgalP-ptrc (example 1E). Both KLpts7/pMCGG and KlgalP-ptrc weretransformed by standard procedures (Sambrook et al., supra) with aplasmid carrying the pathways for glycerol and 1,3-propanediolproduction. The production of cell mass, glycerol and 1,3-propanediolwas tested in fermentations.

A standard fermentation was carried out as follows: A 500 ml seed flaskwas grown at 35° C. in standard 2YT medium (Sambrook et al. supra) for4-6 hours with shaking at 200 rpm. This seed culture was used toincubate a 14 L fermentor which was run in glucose excess conditions at35° C., pH 6.8, for 60 h in a TN2 medium consisting of—

(g/L): K₂HPO₄ (13.6); KH₂PO₄ (13.6): MgSO₄.7H₂O (2); citric acidmonohydrate (2), ferric ammonium citrate (0.3), (NH₄)₂SO₄ (3.2) yeastextract (5) solution of trace elements (1 ml) and pH adjusted to 6.8.The solution of trace elements contained (g/L) citric acid. H₂O (4.0),MnSO₄. H₂O (3.0), NaCl (1.0), FeSO₄.7H₂O (0.10), CoCL₂.6H₂O (0.10),ZnSO₄.7H₂O (0.10), CuSO₄.5H₂O (0.01), H₃BO₃ (0.01) and Na₃MoO₄.2H₂O(0.01)The fermentation was analyzed for cell density by determining theoptical density (OD) of the culture at 600 nM in a spectrophotometer andglycerol and 1,3-propanediol concentrations were determined using HPLC.Isolation and Identification of 1,3-propanediol:

The conversion of glucose to glycerol and 1,3-propanediol was monitoredby HPLC. Analyses were performed using standard techniques and materialsavailable to one of skill in the art of chromatography. One suitablemethod utilized a Waters Alliance HPLC system using RI detection.Samples were injected onto a Aminex HPX87H column (7.8 mm×300 mm,BioRad, Hercules, Calif.) equipped with a Cation H Refill Cartridgeprecolumn (4.6 mm×30 mm, Biorad, Hercules, Calif.), temperaturecontrolled at 50° C., using 5 mM H₂SO₄ as mobile phase at a flow rate of0.4 ml/min. The system was calibrated weekly against known concentrationstandards. Typically, the retention times of glucose, glycerol,1,3-propanediol, and acetate were 12.7 min, 19.0 min, 25.2 min, and 21.5min, respectively.

In this example, two systems were compared. In the first system, the trcpromoter was integrated into the galP target site (galP locus-seeexample 1E) allowing the E. coli strain to produce glucokinase under thenatural regulation of glk and in the second system, the trc promoter wasintegrated into both the galP target site and the glk target site (glkloci—see example 1F). FIGS. 10 and 11 illustrate the fermentationresults for KLpts7/pMCGG and KLgaIP-ptrc transformed with pSYCO101 andpSYCO103, respectively.

The plasmid encoded glucose transport system in KLpts7/pMCGG allowed thestrain to grow to high cell density (FIG. 10A) and produce glycerol and1,3-propanediol (FIG. 10B). However, the amount of glycerol and1,3-propanediol produced relative to the cell mass was low approx 7 g/Lof 1,3 propanediol for an OD₆₀₀ of 194. In contrast, as shown in FIG.11, KlgalP-ptrc produced much less cell mass in the fermentation andmore product approx. 61 g/L 1,3-propanediol and 36 g/L glycerol (FIG.11B) for an OD₆₀₀ of 70 (FIG. 11A).

By constitutively expressing galP on the chromosome from the trcpromoter the flux of carbon from glucose was increased into the pathwayfor the desired products, glycerol and 1,3-propanediol rather than intopathways to produce cell mass.

Example 3 Constitutive Expression of galP and glk from the Chromosome ofa PTS⁻ Strain

The PTS⁻/Glu⁻ strain, KLpts7 was made Glu⁺ by integration of the trcpromoter at the galP and glk target sites to create strain KLGG andreference is made to example 1 above. The strain was transformed bystandard procedures with pSYCO101. The production of cell mass, glyceroland 1,3-propanediol was tested in a standard fermentation (see example2).

As illustrated in FIGS. 11 and 121 in comparison to KlgalP-trc, KLGGgrew more rapidly (at T 33.9, KLGG had obtained an OD₆₀₀ of 24.7 whilethe OD₆₀₀ of KlgalP was 19.6). KLGG produced 70 g/L of 1,3-propanediolcompared with 61 g/L produced by strain KlgalP-trc. Additionally, thepeak concentration was reached earlier in the KLGG fermentation (56.8 hcompared to 62.7 hours for KlgalP). The constitutive expression of galPand glk by chromosomal integration of the trc promoter thereforeproduces more 1,3-propanediol in less fermentation time than theconstitutive expression of only galP.

Example 4 Selection and Analysis of a Fast Growing PTS⁻/Glu⁺ Strain ofE. coli

The long lag phase in growth while producing glycerol and1,3-propanediol demonstrated in fermentation studies of the strain KLGG(in example 3 above) was repeatable in shake flask experiments as shownin table 2 below for KLGG at 24 and 48 hours

TABLE 2 Glucose Time OD Consumed Glycerol 1,3- Molar Strain (h) 600(g/L) (g/L) propanediol Yield KLGG 24 6 5.5 2.6 1.5 1.6 KLGG 48 20 219.9 2.7 1.2 FMP 16 16.5 24.6 9.2 3.2 1.1 Molar yield is (molesglycerol + moles 1,3-propanediol/mole glucose consumedTo decrease the fermentation time a fast growing variant of KLGG wasselected by growing KLGG in a fermentor in TM2 and glucose excessconditions at 35° C. pH 6.8. Cells were harvested at early log phase(for example see T31 in FIG. 12A) and plated for isolated colonies on Lagar. Isolated colonies were screened for variants which producedglycerol and 1,3-propanediol when transformed with pSYCO atconcentrations equivalent to KLGG. A variant was identified anddesignated FMP. FMP exhibited a performance equivalent to KLGG butaccomplished the same performance in 16 h of shake flask growth comparedto 48 h for KLGG (table 2).

Shake flasks experiments were done in TM2 with 2% glucose+spectinomycinat a concentration of 50 microgram/ml and B₁₂ at 2 milligram/liter. Anovernight culture of the strain with the pSYCO plasmid was grown inLB+spectinomycin (50 microgram/ml) at 37° C. with shaking at 200 rpm.The shake flasks were inoculated with 200 microliters of the overnightculture (10 mls of culture in 250 ml baffled flask) and grown at 34° C.with shaking at 200 rpm. The cultures were analyzed for cell density(OD₆₀₀) and consumption of glucose and production of glycerol and1,3-propanediol by HPLC. (Reference is made to example 2).

Analysis of the fast growing variant:: The FMP variant was analyzed forglucokinase activity, relative levels of glk mRNA and the gene andpromoter sequence. As shown in table 3 the glucokinase activity of FMPwas increased 3 fold over that of KLGG, from 0.08 units to 0.22 units.This suggests either a mutation in the coding region resulting in a moreactive enzyme or an increase in the amount of enzyme present.

TABLE 3 Glucokinase activity STRAIN (micromoles/min, mg protein in 1.0mL) KLGG 0.083074232 FMP 0.218672457

To test the expression levels, the relative levels of glk mRNA wasdetermined and reference is made to Table 4A and 4B. Using a lightcycler, data is generated in the form of crossing times. The lower thecrossing time, the more mRNA is present. The crossing times for galPwere equivalent in KLGG and FMP indicating similar levels of mRNA inboth strains. The crossing times of glk in FMP was lower than that ofKLGG (15.7 compared to 18.47, respectively) and the ratio of the averagecrossing times was 1.18 (KLGG-g/k:FMP-g/k), which indicated that moreglk mRNA was present in the FMP strain. Samples were tested in duplicate(1 or 2) and the average (Avg.) was taken. Averages were used todetermine the ratios (glk-K/glk-F represents KLGG glk and FMP glk,respectively).

TABLE 4A Crossing Time ga/P g/k rrsH Strain ga/P1 ga/P2 Avg g/k1 g/k2Avg rrsH1 rrsH2 Avg KLGG 18.48 18.42 18.45 18.47 18.46 18.47 11.64 11.6711.66 FMP 17.75 17.72 17.74 15.69 15.7 15.7 11.7 11.7 11.7

TABLE 4B Ratios rrsH Avg./ rrsH Avg./ ga/P-K Avg./ ga/P-K′/ g/k-K/Strain ga/P Avg. g/k Avg. g/k-F Avg. ga/P-F′ g/k-F KLGG 0.63 0.63 1.01.04 1.18 FMP 0.66 0.75 0.88Sequence analysis was done on the KLGG and FMP glk gene and the trcpromoter. No mutations were found in the glk coding sequence of FMP. Thesequence of the trc promoter was determined by amplification by PCR fromthe chromosome of KLGG and FMP using glkB1/glkBC11 primers. Sequencingwas also performed using the primer TrcF (SEQ ID NO. 14) 5′GCTGTGCAGGTCGTAAATCACTGCATAATT 3′A single mutation from G to A was identified in the lac operator of thetrc promoter in FMP as indicated below. TGGAATTGTGAGCGGATAACAATT: wildtype lac operator (KLGG) (SEQ ID NO. 15) TGGAATTGTGAACGGATAACAATT: lacoperator (FMP) (SEQ ID NO. 16)This mutation has been previously described (THE OPERON, Miller, J H andReznikoff, W S eds., 1980, p190-192 and references therein) as one ofthe O^(c) operator constitutive mutations which increases expression ofthe linked gene(s) and decreases the affinity of the operator for thelac repressor. This effectively would increase the transcription of glkfrom the promoter and this was demonstrated by the increase in enzymeactivity. The variant strain grows faster because there is moreglucokinase to phosphorylate the incoming glucose and more G-6-P will bedelivered into central metabolism.

Assays for Glucokinase Were Done Under the Following Conditions:

100 mM Phosphate Buffer pH 7.2, 5 mM MgCl₂, 500 mM NADP, 5 mM ATP, 2Units of Glucose-6-Phosphate Dehydrogenase. This assay detects theconversion of glucose and ATP to glucose-6-phosphate by monitoring theappearance of NADPH₂ in the following scheme:

Light Cycler Determination of relative levels of mRNA of galP, glk andthe 16S rRNA gene (rrsH) as a control in shake flask experiments.—Thestrains KLGG and FMP were grown in 10 mls of TM2+2% glucose to an OD₆₀₀of 20. The cultures were poured directly into liquid nitrogen in a 50 mlconical tube and RNA was purified as described below. The followingprimers were used:

For galP GalP-R1 5′ GTGTCTTCTTCCTGCCAGAC 3′ (SEQ ID NO. 17) GalP-F15′ CCTGCAACAGTACGCCAAG 3′ (SEQ ID NO. 18) For gLk Glk-R15′ CATCTGGTCCATGTCGATAAGC 3′ (SEQ ID NO. 19) Glk-F15′ GCGGTTGTCAGCTTTCACAA 3′ (SEQ ID NO. 20) For rrsH rrsH-F15′ AGCTGGTCTGAGAGGATG 3′ (SEQ ID NO. 21) rrsH-R1 5′ AATTCCGATTAACGCTTGC3′ (SE ID NO. 22)The light cycler reactions were made according to the manufacturer'sprotocol using Lightcycler RNA Amplication Kit SYBR Green I (Roche)adjusted for 10 μl reactions. A total of 500 ng of RNA were used perreaction. The program used was: target temperature 55° C.; incubationtime 10 min., and temperature transition rate 20° C./sec.

The RNA isolation procedure included growing a strain in a shake flaskunder appropriate conditions as specified in example 4 above andharvesting by pipeting 7 to 10 mls directly into liquid nitrogen in 50ml conical tubes. The samples were frozen at −70° C. until ready foruse. In general, standard procedures were used for RNA isolation withthe following initial adjustments: 50 ml tubes of frozen sample wereplaced in a dry ice bucket; 15 ml of phenol:chloroform (1:1) and 1.5 mlof 3M NaOAc pH 4.8 were added to each 50 ml tube; a small amount of thefrozen sample (ca 500 to 2000 μl of broth) was added to a pre-chilled(with dry ice) coffee grinder; additional dry ice (2 or 3 small pieces)was added to the coffee grinder and samples were ground for at least 1min; the grinder was tapped to get all material into the grinder cap,and the cap, which contained the frozen ground cell broth and residualdry ice, was removed; an equal amount of 2×RNA extraction buffer wasquickly pipetted into the cap; frozen material was stirred into a slurryusing a disposable sterile loop and then placed into conical tubescontaining 15 ml phenol:chloroform/NaOAc; mixed and placed on ice.Standard procedures known in the art were then followed. (Sambrook etal., supra).

1. A method of increasing carbon flow into a metabolic pathway of aPTS⁻/Glu⁻ bacterial host cell which was originally capable of utilizinga phosphotransferase transport system (PTS) for carbohydrate transportcomprising, a) modifying an endogenous chromosomal regulatory regionwhich is operably linked to a nucleic acid encoding a glucoseassimilation protein in a PTS⁻/Glu⁻ host cell by transforming thePTS⁻/Glu⁻ host cell with a DNA construct comprising a promoter and DNAflanking sequences corresponding to upstream (5′) regions of the glucoseassimilation protein; b) allowing integration of the DNA construct torestore a Glu+ phenotype; and c) culturing the transformed host cellunder suitable culture conditions, wherein the carbon flow into ametabolic pathway of the transformed host cell is increased compared tothe carbon flow into the same metabolic pathway in a corresponding PTSbacterial host cell cultured under essentially the same cultureconditions.
 2. The method according to claim 1, wherein the promoter isa non-host cell promoter.
 3. The method according to claim 1, whereinthe promoter is a modified endogenous promoter.
 4. The method accordingto claim 1, wherein the glucose assimilation protein is a glucosetransporter.
 5. The method according to claim 4, wherein the glucosetransporter is a galactose permease obtained from E. coli or a glucosetransporter having at least 80% sequence identity thereto.
 6. The methodaccording to claim 1, wherein the glucose assimilation protein is aphosphorylating protein.
 7. The method according to claim 6, wherein thephosphorylating protein is a glucokinase.
 8. The method according toclaim 5, further comprising modifying an endogenous chromosomalregulatory region which is operably linked to a nucleic acid encoding aglucokinase in the PTS⁻/Glu⁻ host cell by transforming the PTS⁻/Glu⁻host cell with a second DNA construct comprising a promoter and DNAflanking sequences corresponding to upstream (5′) regions of theglucokinase.
 9. The method of claim 1, wherein the bacterial host cellis selected from the group consisting of E. coli cells, Bacillus cellsand Pantoea cells.
 10. The method according to claim 1, wherein thePTS⁻/Glu⁻ host cell is obtained from a PTS cell by deletion of one ormore genes selected from the group consisting of ptsI, ptsH and crr. 11.The method according to claim 1, further comprising transforming thePTS⁻/Glu⁺ host cell with a polynucleotide encoding a protein selectedfrom the group consisting of a transketolase, a transaldolase, and aphosphoenolpyruvate synthase.
 12. The method according to claim 1,further comprising transforming the PTS⁻/Glu⁺ host cell with apolynucleotide encoding at least one enzyme selected from the groupconsisting of DAHP synthase, DHQ synthase, DHQ dehydratase, shikimatedehydrogenase, shikimate kinase EPSP synthase and chorismate synthase.13. The transformed bacterial cell obtained according to the method ofclaim
 1. 14. A method for increasing the production of a desired productin a PTS⁻/Glu⁻ bacterial host cell originally capable of utilizing a PTSfor carbohydrate transport comprising, a) transforming a bacterial hostcell having a PTS⁻/Glu⁻ phenotype with a DNA construct comprising apromoter, wherein said DNA construct is chromosomally integrated intothe PTS⁻/Glu⁻ host cell replacing an endogenous promoter which isoperably linked to a nucleic acid encoding a glucose assimilationprotein; b) culturing the transformed bacterial host cell under suitableconditions; c) allowing expression of the glucose assimilation proteinto obtain a host cell having a PTS⁻/Glu⁺ phenotype; and d) obtaining anincreased amount of a desired product in the transformed bacterial hostcell compared to the amount of the desired product produced in acorresponding PTS bacterial cell cultured under essentially the sameculture conditions, wherein said desired product is selected from thegroup consisting of pyruvate, PEP, lactate, acetate, glycerol, ethanol,succinate and chorismate.
 15. The method according to claim 14, whereinthe host cell is selected from the group consisting of E. coli cells,Bacillus cells and Pantoea cells.
 16. The method according to claim 14,wherein the glucose assimilation protein is a galactose permeaseobtained from E. coli or a glucose transporter having at least 80%sequence identity thereto.
 17. The method according to claim 14, whereinthe glucose assimilation protein is a glucokinase obtained from E. colior a glucokinase having at least 70% sequence identity thereto.
 18. Themethod according to claim 14, wherein the desired product is chorismate.19. The method according to claim 14, wherein the desired product issuccinate.
 20. The method according to claim 14, wherein the desiredproduct is ethanol.
 21. The method according to claim 14, wherein thedesired product is glycerol.
 22. A method of increasing carbon flow intoa metabolic pathway of a PTS⁻/Glu⁻ bacterial host cell originallycapable of utilizing a phosphotransferase transport system (PTS) forcarbohydrate transport comprising, a) modifying an endogenouschromosomal regulatory region which is operably linked to a nucleic acidencoding a galactose permease in a PTS⁻/Glu⁻ host cell by transformingthe PTS⁻/Glu⁻ host cell with a first DNA construct comprising a promoterand DNA flanking sequences corresponding to upstream (5′) regions of thegalactose permease, b) modifying an endogenous chromosomal regulatoryregion which is operably linked to a nucleic acid encoding a glucokinasein the PTS⁻/Glu⁻ host cell by transforming the PTS⁻/Glu⁻ host cell witha second DNA construct comprising a promoter and DNA flanking sequencescorresponding to upstream (5′) regions of the glucokinase; c) allowingintegration of the first and the second DNA constructs, wherein thefirst DNA construct replaces an endogenous promoter of the nucleic acidencoding the galactose permease and the second DNA construct replaces anendogenous promoter of the nucleic acid encoding the glucokinase whereinboth the galactose permease and the glucokinase are expressed in thehost cell and wherein said expression results in an increase in carbonflow into a metabolic pathway of the transformed host cell compared tocarbon flow into the same metabolic pathway in a corresponding unalteredPTS⁻/Glu⁻ bacterial cell.
 23. The method according to claim 22, whereinthe bacterial host cell is selected from the group consisting of E. colicells, Bacillus cells and Pantoea cells.
 24. The method according toclaim 22, wherein the metabolic pathway is the common aromatic pathway.25. The method according to claim 22, further comprising transformingthe PTS⁻/Glu⁻ host cell with a polynucleotide encoding a proteinselected from the group consisting of a transketolase, a transaldolaseand a phosphoenolpyruvate synthase.
 26. A method of restoring a Glu+phenotype to a PTS⁻/Glu⁻ bacterial host cell which was originallycapable of utilizing a phosphotransferase transport system (PTS) forcarbohydrate transport comprising a) modifying an endogenous chromosomalregulatory region which is operably linked to a nucleic acid encoding aglucose transporter in a PTS⁻/Glu⁻ host cell by transforming thePTS⁻/Glu⁻ host cell with a first DNA construct comprising a promoter andDNA flanking sequences corresponding to upstream (5′) regions of theglucose transporter; b) allowing integration of the first DNA construct,wherein the first DNA construct replaces an endogenous promoter of thenucleic acid encoding the glucose transporter; and c) allowingexpression of the glucose transporter, wherein said expression restoresa Glu+ phenotype to the PTS⁻/Glu⁻ host cell.
 27. The method according toclaim 26, wherein the host cell is selected from the group consisting ofE. coli cells, Bacillus cells and Pantoea cells.
 28. The methodaccording to claim 26 further comprising modifying an endogenouschromosomal regulatory region which is operably linked to a nucleic acidencoding a glucokinase in the PTS⁻/Glu⁻ host cell by transforming thePTS⁻/Glu⁻ host cell with a second DNA construct comprising an exogenouspromoter and DNA flanking sequences corresponding to upstream (5′)regions of the glucokinase; allowing integration of the second DNAconstruct wherein the second DNA construct replaces an endogenouspromoter of the nucleic acid encoding the glucokinase; and allowingexpression of the glucokinase.
 29. The method according to claim 28,wherein the host cell is selected from the group consisting of E. colicells, Bacillus cells and Pantoea cells.
 30. The method according toclaim 26, wherein the restored Glu⁺ cells have a specific growth rate ofat least about 0.4 hr⁻¹.
 31. The method according to claim 26, whereinthe glucose transporter is a galactose permease.
 32. A bacterial strainhaving the restored Glu⁺ phenotype obtained according to the method ofclaim
 26. 33. A bacterial strain having the restored Glu⁺ phenotypeobtained according to the method of claim
 28. 34. A method of increasingphosphoenolpyruvate (PEP) availability in a bacterial host cellcomprising, a) selecting a bacterial host cell having a PTS⁻/Glu⁻phenotype, wherein the bacterial host was originally capable ofutilizing a phosphotransferase transport system (PTS) for carbohydratetransport; b) modifying an endogenous chromosomal regulatory sequence ofthe selected bacterial host cell comprising transforming said selectedbacterial host cell with a DNA construct comprising a promoter, whereinsaid DNA construct is chromosomally integrated into the selectedbacterial host cell replacing an endogenous promoter which is operablylinked to a nucleic acid encoding a glucose assimilation protein; c)culturing the transformed bacterial host cell under suitable conditions;and d) allowing expression of the glucose assimilation protein to obtainan altered host cell having a PTS⁻/Glu⁺ phenotype, wherein the PEPavailability is increased compared to the PEP availability in acorresponding unaltered PTS bacterial host cell cultured underessentially the same culture conditions.
 35. The method according toclaim 34, wherein the glucose assimilation protein is a galactosepermease and the DNA construct comprises an exogenous promoter whichreplaces the endogenous promoter of the galactose permease.
 36. Themethod according to claim 34, wherein the glucose assimilation proteinis a glucokinase and the DNA construct comprises an exogenous promoterwhich replaces the endogenous promoter of a glucokinase.
 37. The methodaccording to claim 35 further comprising modifying an endogenouschromosomal regulatory sequence of the selected bacterial host cellcomprising transforming said selected bacterial host cell with a DNAconstruct comprising a promoter, wherein said DNA construct ischromosomally integrated into the selected bacterial host cell replacingan endogenous promoter which is operably linked to a nucleic acidencoding a glucokinase.
 38. The method according to claim 34, whereinthe bacterial host cell is an E. coli cell, a Bacillus cell or a Pantoeacell.
 39. The method according to claim 34 further comprisingtransforming the selected bacterial host cell with a nucleic acidencoding a transketolase, a transaldolase or a phosphoenolpyruvatesynthase.
 40. The altered host cell obtained according to the method ofclaim
 34. 41. A method for increasing the growth rate of a PTS⁻/Glu⁻bacterial host cell originally capable of utilizing a phosphotransferasetransport system (PTS) for carbohydrate transport comprising, a)modifying an endogenous chromosomal regulatory region which is operablylinked to a nucleic acid encoding a galactose permease in a PTS⁻/Glu⁻host cell by transforming the PTS⁻/Glu⁻ host cell with a first DNAconstruct comprising an exogenous promoter and DNA flanking sequencescorresponding to (5′) upstream region of the galactose permease; b)modifying an endogenous regulatory region which is operably linked to anucleic acid encoding a glucokinase in the PTS⁻/Glu⁻ host cell bytransforming the PTS⁻/Glu⁻ host cell with a second DNA constructcomprising an exogenous promoter and DNA flanking sequencescorresponding to upstream (5′) regions of the glucokinase; c) allowingintegration of the first and the second DNA constructs, wherein thefirst DNA construct replaces the endogenous promoter of the nucleic acidencoding the galactose permease and the second DNA construct replacesthe endogenous promoter of the nucleic acid encoding the glucokinase d)culturing the transformed bacterial host cell under suitable conditions;and e) allowing expression of the galactose permease and the glucokinasefrom the modified regulatory regions to obtain an altered bacterial cellhaving an increase specific growth rate compared to the specific growthrate of a corresponding unaltered PTS bacterial host cell cultured underessentially the same culture conditions.
 42. The altered bacterial cellobtained according to the method of claim
 41. 43. The altered bacterialcell of claim 41, wherein said bacterial cell is a Pantoea cell.
 44. Thealtered bacterial cell of claim 41, wherein said bacterial cell is an E.coli cell.
 45. The method according to claim 41 further comprisingtransforming the selected bacterial host cell with a polynucleotideencoding a protein selected from the group consisting of atransketolase, a transaldolase and a phosphoenolpyruvate synthase.
 46. Amethod for increasing the production of a desired product in a PTS⁻/Glu⁻E. coli host cell originally capable of utilizing a PTS for carbohydratetransport comprising, a) modifying an endogenous chromosomal regulatoryregion which is operably linked to a nucleic acid encoding a galactosepermease in an E. coli PTS⁻/Glu⁻ cell by transforming the E. coliPTS⁻/Glu⁻ cell with a first DNA construct comprising an exogenouspromoter and DNA flanking sequences corresponding to upstream (5′)regions of the galactose permease; b) modifying an endogenouschromosomal regulatory region which is operably linked to a nucleic acidencoding a glucokinase in the E. coli PTS⁻/Glu⁻ cell by transforming theE. coli PTS⁻/Glu⁻ cell with a second DNA construct comprising anexogenous promoter and DNA flanking sequences corresponding to upstream(5′) regions of the glucokinase; c) culturing the transformed E. coliPTS⁻/Glu⁻ cell under suitable conditions to allow expression of thegalactose permease and expression of the glucokinase; and d) obtainingan increased amount of a desired product in the transformed E. colicells compared to the amount of the desired product in a correspondingPTS⁻/Glu⁻ E. coli cell cultured under essentially the same cultureconditions wherein the desired product is ethanol, chorismate orsuccinate.
 47. The method according to claim 46, wherein the exogenouspromoter is a non-native promoter selected from the group consisting ofGl, trc, tac and derivative promoters thereof.
 48. The E. coli cellsobtained according to the method of claim 46.